Ginseng polysaccharide GP40025-1 as well as preparation method and application thereof
By combining water decoction extraction with fractional alcohol precipitation and chromatography, the problem of complex and heterogeneous structure of ginseng polysaccharide extract was solved, and high-purity GP40025-1 was obtained, which has significant immunomodulatory activity and is suitable for the preparation of products that enhance immunity.
Patent Information
- Application Number
- CN202610025573.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-09
- Publication Date
- 2026-02-10
Smart Images
Figure CN121495014A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, and in particular to ginseng polysaccharide GP40025-1, its preparation method, and its application. Background Technology
[0002] Ginseng (Panax ginseng CA Meyer) is a perennial herb belonging to the Araliaceae family and the Panax genus, and is abundant in the Changbai Mountains region of Jilin Province. According to the *Compendium of Materia Medica*, ginseng is sweet, slightly bitter, and slightly warm in nature, and enters the spleen, lung, heart, and kidney meridians. Ginseng polysaccharides are one of its main pharmacologically active components, possessing various biological activities such as anti-tumor and immunomodulatory effects. Ginseng polysaccharides have broad application prospects, and their potential mechanisms of action are gradually being discovered by researchers. These mechanisms are usually inextricably linked to the structure of the polysaccharides. However, as biological macromolecules, polysaccharides are characterized by complex structures and difficulties in analysis. Furthermore, obtaining polysaccharides with a uniform and stable structure during preparation is a complex and challenging process. The cellulose in the ginseng cell wall intertwines with the ginseng polysaccharides, forming a strong structural barrier that hinders sufficient contact between the solvent and the polysaccharides, making effective extraction difficult. Therefore, extraction methods need further improvement. Summary of the Invention
[0003] The purpose of this invention is to provide ginseng polysaccharide GP40025-1, its preparation method, and its application, so as to solve the problems existing in the prior art.
[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention, a method for preparing ginseng polysaccharide GP40025-1, includes the following steps: (1) Take ginseng slices and decoct them to obtain an extract. After concentrating the extract, add ethanol for primary alcohol precipitation. Add ethanol to the supernatant after primary alcohol precipitation and let it stand for secondary alcohol precipitation. Centrifuge and collect the precipitate to obtain crude ginseng polysaccharide. Remove protein to obtain refined ginseng polysaccharide. (2) Dissolve the refined ginseng polysaccharide in distilled water and load it onto a pre-equilibrated DEAE-52 cellulose column. Perform gradient elution with sodium chloride aqueous solutions of concentrations of 0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M and 0.40 M. Collect fractions in 12 mL / tube. Determine the sugar content in each tube of eluent using the phenol-sulfuric acid method. Plot the elution curve. Combine the characteristic elution segments according to the elution curve. Collect the elution fraction of the 5th chromatographic peak and name it GP40025. (3) Take the GP40025, dissolve it in NaCl aqueous solution to prepare a 5 mg / mL solution, load it onto a Sephadex G-75 gel chromatography column, use 0.01% NaCl aqueous solution as eluent, flow rate 1 mL / min, collect 2 mL per tube, collect 60 tubes, detect its peak shape and retention time by liquid chromatography, collect appropriate elution peaks, desalt it by Sephadex G-15 gel column chromatography, freeze dry under reduced pressure to obtain the ginseng polysaccharide GP40025-1.
[0005] The second technical solution of the present invention is the ginseng polysaccharide GP40025-1 prepared by the preparation method.
[0006] The third technical solution of the present invention is the application of the ginseng polysaccharide GP40025-1 in the preparation of products that enhance immunity.
[0007] Based on the above technical solution, the present invention has the following technical effects: This invention provides the first-ever isolation and purification of a novel ginseng polysaccharide GP40025-1 from ginseng, exhibiting a uniform structure and a weight-average molecular weight of 116,240 g / mol. Its monosaccharide composition comprises mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in a molar ratio of 5.88:10.64:17.49:11.35:40.72:7.35:6.57. This polysaccharide possesses a well-defined chemical structure and a unique sugar chain linkage, thus solving the technical challenges of complex and heterogeneous structures and difficulties in standardized quality control of existing ginseng polysaccharide extracts.
[0008] The preparation method established in this invention employs a systematic purification process combining water decoction extraction, fractional alcohol precipitation for impurity removal, DEAE-52 cellulose ion exchange chromatography, and Sephadex G-75 gel filtration chromatography. This process effectively removes proteins, pigments, and other heteropolysaccharides, yielding high-purity, structurally stable target polysaccharide components. The process exhibits good reproducibility and stable yield, providing a reliable technical solution for the large-scale preparation and industrial application of ginseng polysaccharides.
[0009] Pharmacological activity studies have shown that the ginseng polysaccharide GP40025-1 provided by this invention can significantly promote the proliferation of mouse macrophages RAW264.7 and effectively improve the phagocytic capacity of the cells in a dose-dependent manner within the concentration range of 12.5-200 μg / mL, demonstrating excellent immunomodulatory activity. It has broad application prospects in the preparation of drugs, functional foods or health products that enhance immunity. Attached Figure Description
[0010] Figure 1 Elution curves for refined ginseng polysaccharides.
[0011] Figure 2 The effect of ginseng polysaccharide GP40025-1 on the proliferation of RAW264.7 cells.
[0012] Figure 3 The effect of ginseng polysaccharide GP40025-1 on the phagocytic rate of RAW264.7 cells.
[0013] Figure 4 The effect of ginseng polysaccharide GP40015-1 on the proliferation of RAW264.7 cells.
[0014] Figure 5 The effect of ginseng polysaccharide GP40015-1 on the phagocytic rate of RAW264.7 cells.
[0015] Figure 6 The effect of ginseng polysaccharide GP40040-1 on the proliferation of RAW264.7 cells.
[0016] Figure 7 The effect of ginseng polysaccharide GP40040-1 on the phagocytic rate of RAW264.7 cells.
[0017] Figure 8 GC-MS image of ginseng homogeneous polysaccharide GP40025-1 after methylation analysis.
[0018] Figure 9 GP40025-1 1 H-NMR spectrum.
[0019] Figure 10 GP40025-1 13 C-NMR spectrum.
[0020] Figure 11 GP40025-1 1 H- 1 H COSY spectrum.
[0021] Figure 12 This is the HSQC spectrum of GP40025-1.
[0022] Figure 13 The spectrum is GP40025-1 HMBC.
[0023] Figure 14 for m / z 281.3 Oligosaccharide fragments in negative ion mode ESI-MS 2 Mass spectrum.
[0024] Figure 15 for m / z 281.3 Structural formulas and cleavage mechanisms of oligosaccharide fragments.
[0025] Figure 16for m / z 339.3 ESI-MS of oligosaccharide fragments in negative ion mode 2 Mass spectrum.
[0026] Figure 17 for m / z 339.3 Structural formula and cleavage mechanism of oligosaccharide fragments.
[0027] Figure 18 for m / z 355.2 Oligosaccharide fragments in negative ion mode ESI-MS 2 Mass spectrum.
[0028] Figure 19 for m / z 355.2 Structural formulas and cleavage mechanisms of oligosaccharide fragments.
[0029] Figure 20 for m / z 369.4 ESI-MS of oligosaccharide fragments in negative ion mode 2 Mass spectrum.
[0030] Figure 21 for m / z 369.4 Structural formulas and cleavage mechanisms of oligosaccharide fragments.
[0031] Figure 22 for m / z 503.5 ESI-MS of oligosaccharide fragments in negative ion mode 2 Mass spectrum.
[0032] Figure 23 for m / z 503.5 Structural formulas and cleavage mechanisms of oligosaccharide fragments.
[0033] Figure 24 for m / z 665.3 ESI-MS of oligosaccharide fragments in negative ion mode 2 Mass spectrum.
[0034] Figure 25 for m / z 665.3 Structural formulas and cleavage mechanisms of oligosaccharide fragments. Detailed Implementation
[0035] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0036] This invention provides a method for preparing ginseng polysaccharide GP40025-1, comprising the following steps: (1) Take ginseng slices and decoct them to obtain an extract. After concentrating the extract, add ethanol for primary alcohol precipitation. Add ethanol to the supernatant after primary alcohol precipitation and let it stand for secondary alcohol precipitation. Centrifuge and collect the precipitate to obtain crude ginseng polysaccharide. Remove protein to obtain refined ginseng polysaccharide. This invention innovatively uses multiple alcohol precipitation, that is, first precipitate alcohol to discard the polysaccharide part with a larger molecular weight, then add ethanol to precipitate to obtain the polysaccharide part with a middle molecular weight, and discard the polysaccharide part with a smaller molecular weight.
[0037] (2) Dissolve the refined ginseng polysaccharide in distilled water and load it onto a pre-equilibrated DEAE-52 cellulose column. Perform gradient elution with sodium chloride aqueous solutions of concentrations of 0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M and 0.40 M. Collect fractions in 12 mL / tube. Determine the sugar content in each tube of eluent using the phenol-sulfuric acid method. Plot the elution curve. Combine the characteristic elution segments according to the elution curve. Collect the elution fraction of the 5th chromatographic peak and name it GP40025. (3) Take the GP40025, dissolve it in NaCl aqueous solution to prepare a 5 mg / mL solution, load it onto a Sephadex G-75 gel chromatography column, use 0.01% NaCl aqueous solution as eluent, flow rate 1 mL / min, collect 2 mL per tube, collect 60 tubes, detect its peak shape and retention time by liquid chromatography, collect appropriate elution peaks, desalt it by Sephadex G-15 gel column chromatography, freeze dry under reduced pressure to obtain the ginseng polysaccharide GP40025-1.
[0038] In some specific implementation plans, the weight ratio of ginseng slices to water is 1:10; The conditions for concentrating the extract are to concentrate it to one-fifth to one-tenth of its volume.
[0039] In some specific implementations, the primary alcohol precipitation method is as follows: add 95% ethanol by volume until the ethanol concentration in the system reaches 20% (v / v), and let it stand at room temperature for 12 hours.
[0040] In some specific implementations, the conditions for the second ethanol precipitation are as follows: add 95% ethanol by volume to a final ethanol concentration of 40% (v / v) and let stand at room temperature for 12 h.
[0041] In some specific implementations, the ratio of the refined ginseng polysaccharide to distilled water is 4.0 g: 30 mL.
[0042] This invention also provides ginseng polysaccharide GP40025-1 prepared by the aforementioned preparation method.
[0043] In some specific implementation schemes, the structure is as follows: .
[0044] This invention also provides the application of the ginseng polysaccharide GP40025-1 in the preparation of products that enhance immunity.
[0045] Example 1 1. Preparation of Ginseng Polysaccharides Ginseng slices were decocted twice with 10 times their weight of water, each time for 1.5 hours. The two decoctions were combined to obtain an extract. The extract was concentrated (to one-fifth of its volume), and 95% ethanol was added until the ethanol concentration reached 20% (v / v). The mixture was allowed to stand at room temperature for 12 hours to complete primary ethanol precipitation. The mixture was centrifuged (3000 rpm for 10 minutes). The supernatant after primary ethanol precipitation was added to 95% ethanol until the final ethanol concentration reached 40% (v / v). The mixture was allowed to stand at room temperature for 12 hours, and then centrifuged (3000 rpm for 10 minutes) to collect the precipitate. Crude ginseng polysaccharide was obtained. Protein was removed using the Sevag method to obtain purified ginseng polysaccharide.
[0046] 4.0 g of purified ginseng polysaccharide sample was weighed, dissolved in 30 mL of distilled water, and loaded onto a pre-equilibrated DEAE-52 cellulose column at a slow flow rate. Gradient elution was performed using sodium chloride aqueous solutions of different concentrations (0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, and 0.40 M). Fractions were collected in 12 mL fractions per tube. The sugar content in each eluent was determined using the phenol-sulfuric acid method. Elution curves were plotted, and characteristic elution segments were combined based on the elution curves. The elution fraction from the 5th chromatographic peak, GP40025, was collected. The elution curve is shown below. Figure 1 As shown.
[0047] Ginseng polysaccharide fraction GP40025 was dissolved in 0.10 M NaCl aqueous solution to prepare a 5 mg / mL solution. The solution was loaded onto a Sephadex G-75 gel chromatography column (3.0 × 90 cm). The eluent was 0.01% NaCl aqueous solution, the flow rate was 1 mL / min, and 2 mL was collected from each tube for 60 tubes. The peak shape and retention time were detected by liquid chromatography, and appropriate elution peaks were collected. After desalting by Sephadex G-15 gel column chromatography, the solution was lyophilized under reduced pressure to obtain the new ginseng polysaccharide GP40025-1.
[0048] 2. Chemical characteristics of ginseng polysaccharides Analysis showed that the weight-average molecular weight of ginseng polysaccharide GP40025-1 was 116240 g / mol, mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in a ratio of 5.88:10.64:17.49:11.35:40.72:7.35:6.57. No other ginseng polysaccharides with the same chemical characteristics have been reported to date.
[0049] 3. Pharmacological activities of ginseng polysaccharides RAW264.7 phagocytes from mice in the logarithmic growth phase and in good growth condition were selected, washed three times with PBS buffer, and then the cell suspension density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 10,000 cells / mL. Then, 100 μL of cell suspension was added to each well of a 96-well plate to ensure a cell count of 10,000 per well. According to the experimental design, cells were seeded into different wells of the 96-well plate. The 96-well plates were then incubated at 37°C with 5% CO2 for 24 h. After incubation, the supernatant was discarded. A control group (cells present but no drug intervention) was established; a blank group (DMEM medium containing only 10% fetal bovine serum with no cells) was also established; and a graded drug intervention group (cells present and drug intervention administered). The graded drug intervention groups were given different concentrations of ginseng homogenized polysaccharide sample solution: 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, with six replicates for each concentration. The plates were then incubated at 37°C for 24 h. After 24 h of incubation, 10 µL of CCK-8 solution was added to each well, and the samples were incubated in a CO2 incubator for another 2 h. The absorbance (OD value) of the samples was measured at 450 nm using a microplate reader and calculated according to the following formula: Cell proliferation rate = (A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 ) × 100%.
[0050] This invention investigated the pharmacological activity of ginseng polysaccharide GP40025-1, and the results suggest that this polysaccharide has a pharmacological effect of regulating the activity of RAW264.7 cells. The results indicate that ginseng polysaccharide can promote the proliferation of RAW264.7 cells, as shown below. Figure 2 As shown.
[0051] Cells in the logarithmic growth phase were collected and counted using a cell counter. The concentration of the cell suspension was adjusted to achieve a density of 1 × 10⁻⁶. 5Cells / mL were collected in 96-well plates, with blank control, positive control, and experimental groups (5 drug concentrations). In the control and experimental groups, 200 μL of cell suspension was added to each well; while in the blank group, 200 μL of complete culture medium was added to each well. Each group had 6 replicates and was incubated at 37°C for 24 h. After 24 h, the old medium was discarded. 200 μL of fresh complete culture medium was added to the control and blank groups respectively; LPS was added to the control group, while 200 μL of the test sample solution containing different concentrations (25 μg / mL, 57 μg / mL, 177 μg / mL, 277 μg / mL, 477 μg / mL, 877 μg / mL) was added to the experimental groups, and incubation continued for 24 h. The old medium was discarded, and 200 μL of PBS containing neutral red was added to each well, incubated at 37°C for 1 h. Afterwards, remove the PBS solution containing neutral red and wash 1-2 times with PBS preheated to 37°C. Then, add 200 μL of neutral red detection lysis buffer to each well and lyse on a shaker at room temperature for 10 min. Measure the OD value at 540 nm using a microplate reader and calculate according to the following formula: Attack rate = (A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 ) × 100%.
[0052] Ginseng polysaccharide GP40025-1 can increase the phagocytic rate of RAW264.7 cells, as shown in the results. Figure 3 As shown.
[0053] Comparative Example 1 1. Preparation of Ginseng Polysaccharides Ginseng slices were decocted twice with 10 times their weight of water, each time for 1.5 hours. The two decoctions were combined to obtain an extract. The extract was concentrated (to one-fifth of its volume), and 95% ethanol was added until the ethanol concentration reached 20% (v / v). The mixture was allowed to stand at room temperature for 12 hours to complete primary ethanol precipitation. The mixture was centrifuged (3000 rpm for 10 minutes). The supernatant after primary ethanol precipitation was added to 95% ethanol until the final ethanol concentration reached 40% (v / v). The mixture was allowed to stand at room temperature for 12 hours, and then centrifuged (3000 rpm for 10 minutes) to collect the precipitate. Crude ginseng polysaccharide was obtained. Protein was removed using the Sevag method to obtain purified ginseng polysaccharide.
[0054] Weigh 4.0 g of purified ginseng polysaccharide sample, dissolve it in 30 mL of distilled water, and load it onto a pre-equilibrated DEAE-52 cellulose column at a slow flow rate. Perform gradient elution with different concentrations of sodium chloride aqueous solution (0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, and 0.40 M). Collect fractions at 12 mL / tube. Determine the sugar content in each eluent using the phenol-sulfuric acid method. Plot the elution curve, combine the characteristic elution segments according to the elution curve, and collect the eluent fraction of the third chromatographic peak: GP40015.
[0055] Ginseng polysaccharide fraction GP40015 was dissolved in 0.10 M NaCl aqueous solution to prepare a 5 mg / mL solution. The solution was loaded onto a Sepharose CL-6B gel chromatography column (3.0 × 90 cm). The eluent was 0.01% NaCl aqueous solution, the flow rate was 1 mL / min, and 2 mL was collected from each tube for 60 tubes. The peak shape and retention time were detected by liquid chromatography, and appropriate elution peaks were collected. After desalting by Sephadex G-15 gel column chromatography, the solution was lyophilized under reduced pressure to obtain the new ginseng polysaccharide GP40015-1.
[0056] 2. Chemical characteristics of ginseng polysaccharides Analysis showed that the weight-average molecular weight of ginseng polysaccharide GP40015-1 was 2,123,789 g / mol, mainly composed of 1-glucose, 1,4-glucose, 1,6-glucose, 1,3-glucose, and 1,3,4-glucose in a ratio of 14.3:46.2:10.4:20.2:8.9. No other ginseng polysaccharides with the same chemical characteristics have been reported to date.
[0057] 3. Pharmacological activities of ginseng polysaccharides The pharmacological activity of ginseng polysaccharide GP40015-1 was investigated, and the results suggested that this polysaccharide has a pharmacological effect on regulating the activity of RAW264.7 cells. The results showed that ginseng polysaccharide can promote the proliferation of RAW264.7 cells, as indicated by the following results. Figure 4 As shown.
[0058] Ginseng polysaccharide GP40015-1 can increase the phagocytic rate of RAW264.7 cells, as shown in the results. Figure 5 As shown.
[0059] Comparative Example 2 1. Preparation of Ginseng Polysaccharides Ginseng slices were decocted twice with 10 times their weight of water, each time for 1.5 hours. The two decoctions were combined to obtain an extract. The extract was concentrated (to one-fifth of its volume), and 95% ethanol was added until the ethanol concentration reached 20% (v / v). The mixture was allowed to stand at room temperature for 12 hours to complete primary ethanol precipitation. The mixture was centrifuged (3000 rpm for 10 minutes). The supernatant after primary ethanol precipitation was added to 95% ethanol until the final ethanol concentration reached 40% (v / v). The mixture was allowed to stand at room temperature for 12 hours, and then centrifuged (3000 rpm for 10 minutes) to collect the precipitate. Crude ginseng polysaccharide was obtained. Protein was removed using the Sevag method to obtain purified ginseng polysaccharide.
[0060] Weigh 4.0 g of purified ginseng polysaccharide sample, dissolve it in 30 mL of distilled water, and load it onto a pre-equilibrated DEAE-52 cellulose column at a slow flow rate. Perform gradient elution with different concentrations of sodium chloride aqueous solution (0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, and 0.40 M). Collect fractions at 12 mL / tube. Determine the sugar content in each eluent using the phenol-sulfuric acid method. Plot the elution curve, combine the characteristic elution segments according to the elution curve, and collect the elution fraction of the 6th chromatographic peak: GP40040.
[0061] Ginseng polysaccharide fraction GP40040 was dissolved in 0.10 M NaCl aqueous solution to prepare a 5 mg / mL solution. The solution was loaded onto a Sephadex G-75 gel chromatography column (3.0 × 90 cm). The eluent was 0.01% NaCl aqueous solution. The flow rate was 1 mL / min. 2 mL was collected from each tube, and 60 tubes were collected. The peak shape and retention time were detected by liquid chromatography. Appropriate elution peaks were collected. After desalting by Sephadex G-15 gel column chromatography, the solution was lyophilized under reduced pressure to obtain the new ginseng polysaccharide GP40040-1.
[0062] 2. Chemical characteristics of ginseng polysaccharides Analysis showed that the weight-average molecular weight of ginseng polysaccharide GP40040-1 was 230,857 g / mol, mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in a ratio of 5.55:18.22:34.50:15.54:10.24:6.21:9.74. No other ginseng polysaccharide with the same chemical characteristics has been reported to date.
[0063] 3. Pharmacological activities of ginseng polysaccharides This invention investigated the pharmacological activity of ginseng polysaccharide GP40040-1, and the results suggest that this polysaccharide has a pharmacological effect of regulating the activity of RAW264.7 cells. The results indicate that ginseng polysaccharide can promote the proliferation of RAW264.7 cells, as shown below. Figure 6 As shown.
[0064] Ginseng polysaccharide GP40040-1 can increase the phagocytic rate of RAW264.7 cells, as shown in the results. Figure 7 As shown.
[0065] Example 2 Structural analysis of ginseng polysaccharide GP40025-1: 1 GP40025-1 methylation The main steps include: (1) Full methylation modification: In polysaccharide methylation experiments, the dried polysaccharide sample must first be dissolved in DMSO (dimethyl sulfoxide). To avoid moisture interference with the experimental results, this operation is usually carried out under an inert atmosphere such as nitrogen or argon. After dissolution, an alkaline substance such as sodium hydroxide or alkaline carbonate is added to the solution. Then, methyl iodoform (CH3I) is added. Methyl iodoform reacts with the activated free hydroxyl groups, methylating them and ultimately forming methoxy groups. This reaction is carried out at room temperature, and the reaction time varies from several hours to overnight, in order to ensure that all free hydroxyl groups are fully methylated.
[0066] (2) Hydrolysis: The methylated polysaccharide needs to be hydrolyzed by trifluoroacetic acid to break the glycosidic bond and generate monosaccharide units.
[0067] (3) Reduction: Use the reducing agent NaBH4 to reduce monosaccharides to sugar alcohols.
[0068] (4) Acetylation: The reduced sugar alcohol is acetylated to generate an acetate derivative. Acetylation helps improve the volatility and stability of subsequent GC-MS analysis.
[0069] (5) Gas chromatography-mass spectrometry (GC-MS) analysis: By analyzing the peak retention time of the gas chromatography and the main ion fragments of the mass spectrum, the linkage type of sugar can be determined more accurately.
[0070] The homogeneous polysaccharide GP40025-1 from ginseng underwent three methylation treatments, followed by detection and analysis using infrared spectroscopy (IR). At 3300 cm⁻¹... -1 At this point, the originally strong and broad -OH vibration absorption peak basically disappeared; at the same time, a peak at 2900 cm⁻¹ was observed. -1 The vibrational absorption peak of the nearby methyl group is significantly enhanced, and from 1000 to 1400 cm⁻¹ -1The CO vibrational absorption peaks within the wavelength range also showed a significant increasing trend. These changes in infrared spectral characteristics indicate that the polysaccharide sample has reached a fully methylated state. The methylated products were analyzed using GC-MS. During the analysis, the obtained data were compared with standard mass spectrometry libraries and references. The relative retention times of different sugar alcohol acetyl esters in gas chromatography (e.g., ...) were compared. Figure 8 As shown in the figure, the methylation analysis results were obtained and summarized in Table 1.
[0071] The results showed that GP40025-1 has a relatively complex branched structure. The edge of the molecule is mainly Ara, and the interior of GP40025-1 contains various branched sugar residues such as 1,3,4-GalA, 1,2,4-GalA, 1,4,6-Man, 1,2,4-Gal, and 1,3,6-Man.
[0072] Table 1. Methylation analysis results of ginseng homogeneous polysaccharide GP40025-1
[0073] 2 GP40025-1 NMR Analysis Approximately 20 mg of dried ginseng homogenized polysaccharide sample was weighed and placed in a container. 0.6 mL of D2O solvent (99.8% purity) was added, and the mixture was stirred thoroughly to ensure complete dissolution. The resulting solution was then transferred to an NMR tube and analyzed on a Bruker AV700 MHz NMR spectrometer. 13 C-NMR, 1 H-NMR, HH COSY, HSQC, and HMBC spectra were used to obtain relevant structural information.
[0074] Based on the composition of monosaccharides, methylation analysis data obtained by GC-MS, 1D and 2D NMR spectral information, and referring to relevant literature reports, we analyzed GP40025-1... 1 H NMR and 13 A comprehensive assignment analysis was performed on the C10 NMR spectra. Most of them... α - Anisotropic protons appear at 100-102.5 ppm. For example, GP40025-1 contains →6)Man(1→, →2)Man(1→, →3)Glc(1→, →4)Man(1→ residues, with chemical shift values of 99.1, 102.3, 102.0, and 95.2, respectively. Based on comparison with literature, its configuration is inferred to be →6). α -Man(1→,→2) α -Man(1→,→3) α -Glc(1→,→4) α-Man (1→, as can be seen from the figure, 102.2, 103.0, and 103.5 are →2) β -Ara(1→,→3) β -Ara(1→,→3,4) β -Ara(1→)
[0075] In ginseng homogeneous polysaccharide GP40025-1 1 In the H-NMR spectrum (see) Figure 9 ), δ H The range of 5.50 to 4.90 is attributed to the H-1 terminal hydrogen signal of sugars. δ H The range of 4.80 to 3.30 is attributed to non-terminal hydrogen signals of sugars; δ H 5.11 is →2)- β -Ara(1→H-1 signal, δ H 5.04 is →3)- β -Ara(1→H-1 signal, δ H 5.41 is β -Rha(1→H-1 signal, δ H 4.89 is →2,3)- β -Ara(1→H-1 signal, δ H 4.60 is → 6) α -Man(1→H-1 signal, δ H 5.09 is →2) α -Man(1→H-1 signal, δ H 5.09 is →3)- β -Man(1→H-1 signal, δ H 5.10 is →3)- α -Glc(1→H-1 signal, δ H 4.64 is →4)- β -GlcA(1→H-1 signal, δ H 5.30 is →4)- α -Man(1→H-1 signal, δ H 4.96 is →2,3)- β -GalA(1→H-1 signal, δ H5.08 is →2,4)- β -GalA(1→H-1 signal, δ H 4.90 is → 4,6) - β -Man(1→H-1 signal, δ H 4.97 is → 4,6) - β -Gal(1→H-1 signal).
[0076] In ginseng homogeneous polysaccharide GP40025-1 13 In the C-NMR spectrum (see) Figure 10 ), δ c102.2 is →2)- β -Ara(1→C-1 signal, δ c103.0 is →3)- β -Ara(1→C-1 signal, δ c102.4 is β -Rha(1→C-1 signal, δ c103.5 is →2,3)- β -Ara(1→C-1 signal, δ c99.1 is →6)- α -Man(1→C-1 signal, δ c102.3 is →2) α -Man(1→C-1 signal, δ c110.4 is →3)- β -Man(1→C-1 signal, δ c102.0 is →3)- α -Glc(1→C-1 signal, δ c107.2 is →4)- β -GlcA(1→C-1 signal, δ c95.2 is →4)- α -Man(1→C-1 signal, δ c103.3 is →2,3)- β -GalA(1→C-1 signal, δ c110.4 is →2,4)- β -GalA(1→C-1 signal, δ c103.1 is →4,6)- β -Man(1→C-1 signal, δ c103.2 is →4,6)- β -Gal(1→C-1 signal).
[0077] Combined with the HH COSY and HSQC spectra of ginseng homogeneous polysaccharide GP40025-1 ( Figure 11-12 The remaining signals of these residues were fully assigned based on the results of COSY and HSQC, and are summarized in Table 2.
[0078] Table 2. Attribution of HSQC correlation peak signals of ginseng homogeneous polysaccharide GP40025-1
[0079] Application of HMBC spectroscopy ( Figure 13 Further analysis of the skeletal structure and substitution sites was conducted. Based on the HMBC results, the remaining signals of these residues were fully assigned and summarized in Table 3.
[0080] Table 3. HMBC key correlation peak signal assignment and sugar residue linkage relationship of GP40025-1
[0081] 3. Analysis of GP40025-1 enzymatic digest fragments Enzymatic hydrolysis, through the precise disassembly of polysaccharide molecules, has become a core method for revealing polysaccharide structures. Given the differences in the composition of ginseng polysaccharides at each stage, different enzymes need to be selected to explore their structural characteristics. 1 mg of homogeneous ginseng polysaccharide GP40025-1 was accurately weighed and placed in a container. Then, 1 mL of distilled water was added to the container, and the mixture was stirred thoroughly to completely dissolve the polysaccharide sample. After complete dissolution, 25 U of cellulase was added to the container. The container was placed at 40°C for 1 h of hydrolysis. After the hydrolysis reaction was complete, the reaction mixture was heated in boiling water for 10 min, and then centrifuged to remove inactivated cellulase, preparing for subsequent analysis of the hydrolysis products.
[0082] Systematic characterization of the enzymatic hydrolysis products of ginseng homogeneous polysaccharide GP40025-1 was performed. Characteristic oligosaccharide fragments with degrees of polymerization of 2-4 were identified through gradient enzymatic hydrolysis and mass spectrometry screening of oligosaccharide components, with mass-to-charge ratios covering a wide range. m / z Key signals include 281.3 (disaccharide), 339.3 (disaccharide), 355.2 (disaccharide), 369.4 (disaccharide), 503.2 (trisaccharide), 517.1 (branched trisaccharide), 545.2 (trisaccharide), and 665.5 (tetrasaccharide). Multistage mass spectrometry fragmentation mode analysis is detailed in Table 4.
[0083] Table 4. ESI-MS2 analysis results of oligosaccharide fragments in the enzymatic hydrolysis products of ginseng homogeneous polysaccharide GP40025-1.
[0084] Electrospray tandem mass spectrometry (ESI-MS)2 )Graph Analysis ( Figure 14 ), parent ion peak m / z 281.3 was classified as a deprotonated product of the diarabinose unit [Ara-Ara-H]. - (C 10 H 18 The structure of O9) and the collision-induced dissociation process of this disaccharide molecule exhibit multi-dimensional cleavage characteristics: glycosidic bond breakage to form m / z 131.1 [Ara-H2O] - and m / z 149.2 [Ara-H] - A fracture within the ring will form something like... m / z 89.1 ( 1,4 (A1 ring fracture) m / z 221.2 ( 0,2 Characteristic secondary fragments are generated, such as those produced by A2 breakage. This was achieved through synergistic analysis integrating glycosidic bond breakage and transcyclic fragmentation data (oligosaccharide fragment structures and fragmentation mechanisms are shown in [reference]). Figure 15 This clearly indicates that the oligosaccharide fragment is the structural unit Ara-Ara.
[0085] based on Figure 16 Electrospray tandem mass spectrometry (ESI-MS) 2 Data analysis, parent ion peak m / z 339.3 was identified as a deprotonated product of galacturonic acid-rhamnose dimer. m / z 339.3[GalA-Rha-H] - (C 12 H 20 O 11 During collision-induced dissociation, this molecule exhibits a dual-pathway cleavage characteristic: specific cleavage of glycoside linkages to generate... m / z 163.2 rhamnose monomer fragments [Rha-H](C6H) 11 O5) and m / z The galacturonic acid dehydration product [GalA-H2O](C6H9O6) at 177.1 was observed, along with multiple sets of intraring breakage characteristic peaks detected in the spectrum, including... m / z 72.9 ( 0,3 (A1 ring fracture) m / z 103.7 ( 0,2 (A1 ring fracture) m / z 293.4 ( 2,5 Secondary characteristic ions include A2 ring breakage. Synergistic analysis integrating glycosidic bond breakage and transcyclic fragmentation data (oligosaccharide fragment structures and fragmentation mechanisms are shown in [reference]). Figure 17 This clearly indicates that the oligosaccharide fragment is a GalA-Rha structural unit.
[0086] based on Figure 18 Electrospray tandem mass spectrometry (ESI-MS) 2 Analysis of the parent ion peak m / z 355.2 was classified as a deprotonated product of galacturonic acid-glucose dimer [GalA-Glc-H]. - (C 12 H 20 O 12 The structure of the molecule is as follows. During collision-induced dissociation, the glycosidic bond undergoes specific cleavage, generating... m / z 163.0 g of glucose dehydration product [Glc-H2O] - , m / z 179.0 [Glc-H] - and m / z The characteristic galacturonic acid fragment [GalA-H] at 193.1 - The spectrum simultaneously detected multiple sets of intra-ring fracture characteristic peaks, including, for example... m / z 89.1 is 3,5 Fragment ions broken within the A1 ring and m / z 119.0 is 2,4 Fragment ions from the A1 ring breakage. Synergistic analysis of glycosidic bond breakage and intra-ring cleavage modes (oligosaccharide fragment structures and cleavage mechanisms are shown in [reference]). Figure 19 This confirms that the oligosaccharide fragment is a GalA-Glc structural unit.
[0087] based on Figure 20 Electrospray tandem mass spectrometry (ESI-MS) 2 Spectrum analysis, parent ion peak m / z 369.4 was identified as the deprotonated product of digalacturonic acid [GalA-GalA-H]. - (C 12 H 20 O 12 The structure was described. Collision-induced dissociation experiments revealed that the dimer exhibited a two-pathway cleavage characteristic: glycoside linkages were specifically broken to generate... m / z 177.2% glucose dehydration product [Glc-H2O](C6H 11 O6) m / z 193.0 g of galacturonic acid monomer fragment [GalA-H](C6H) 11 O7); Simultaneously, multiple sets of intra-ring fracture characteristic peaks were detected, including m / z 73.2 is ( 0,4 (A1 ring fracture) m / z 104.7 ( 0,3 A1 ring fracture) and m / z 235.0 ( 2,4Secondary characteristic ions include A2 ring breakage. Synergistic analysis of glycosidic bond breakage and transcyclic fragmentation data (oligosaccharide fragment structures and fragmentation mechanisms are shown in...) Figure 21 This clearly indicates that the oligosaccharide fragment is a GalA-GalA structural unit.
[0088] based on Figure 22 Electrospray tandem mass spectrometry (ESI-MS) 2 Data analysis, parent ion peak m / z 503.5 was classified as the deprotonated product of the rhamnose-galacturonic acid-mannose trimer [Rha-GalA-Man-H]. - (C 12 H 20 O 12 The structure of this trisaccharide molecule is as follows: during collision-induced dissociation, the molecule exhibits a step-by-step cleavage pattern: the terminal glycosidic bond breaks to form a glycosidic bond. m / z 146.9 g of rhamnose dehydration product [Rha-H2O] - , m / z 162.4% rhamnose monomer fragments [Rha-H] - and m / z Characteristic fragments of mannose at 179.1 [Man-H] - Simultaneously, multi-level intraring fracture products were detected, including such as m / z 104.9 ( 0,2 (A1 ring fracture) m / z 280.7 ( 0,2 (fracture within A2 ring) m / z 221.2 ( 0,2 X1 ring fracture) and m / z 382.1 ( 2,4 Characteristic secondary ions such as A3 ring breakage. Synergistic verification of over-integration terminal breakage and trans-ring fragmentation data (oligosaccharide fragment structures and fragmentation mechanisms are shown in...). Figure 23 This clearly indicates that the oligosaccharide fragment is a Rha-GalA-Man structural unit.
[0089] based on Figure 24 Electrospray tandem mass spectrometry (ESI-MS) 2 Data analysis, parent ion peak m / z 665.2 was classified as a deprotonated tetraglucose product [Glc-Glc-Glc-Glc-H] - (C 24 H 42 O 21 During collision-induced dissociation, the molecule exhibits a step-by-step cleavage pattern: the terminal glycosidic bonds break sequentially to form... m / z 162.6g of dehydrated glucose product [Glc-H2O] - (C6H11 O5) m / z 179.3 [Glc-H] - (C6H 11 O6) m / z 341.1% diglucan [Glc-Glc-H] - (C 12 H 21 O 11 )and m / z Triglucose fragments [Glc-Glc-Glc-H] at 503.4 - (C 18 H 31 O 16 Multiple sets of transcyclic fracture characteristic peaks were detected simultaneously in the spectrum, including, for example... m / z 220.9 ( 2,4 (fracture within A2 ring) m / z 265.6 ( 2,5 (fracture within A2 ring) m / z 281.4 ( 0,2 (A1 ring fracture) m / z 383.1 ( 2,4 (fracture within A3 ring) m / z 443.2 ( 0,2 (fracture within A3 ring) m / z 545.3 ( 2,4 (A4 ring fracture) and m / z 588.0 ( 2,5 (A4 ring breakage), etc. Synergistic analysis was conducted by integrating step-by-step glycosidic bond breakage and higher-order transcyclic fragmentation data (oligosaccharide fragment structures and fragmentation mechanisms are shown in...). Figure 25 This clearly indicates that the oligosaccharide fragment is a Glc-Glc-Glc-Glc tetrasaccharide structural unit.
[0090] Based on a multi-dimensional structural analysis strategy of enzymatic hydrolysis products, this invention systematically elucidates the molecular structural features of ginseng homogeneous polysaccharide GP40025-1. This is achieved through electrospray ionization tandem mass spectrometry (ESI-MS) integrating the disaccharide components. 2 By combining the fragmentation pattern with NMR anomeric proton shift data, a two-way verification of the glycounit linkage mode was achieved. Further, by combining the specific mass spectrometric fragmentation characteristics of the trisaccharide and tetrasaccharide components (such as transring breakage and stepwise glycosidic bond dissociation), the topological arrangement of complex oligosaccharide sequences in the polysaccharide molecule was successfully deduced. Through the fusion of substitution site analysis from 1H NMR spectroscopy, structural unit verification from controlled hydrolysis experiments, and quantitative data on sugar residue linkage sites provided by methylation analysis, the structure of GP40025-1 was finally deduced as follows: .
[0091] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing ginseng polysaccharide GP40025-1, characterized in that, Includes the following steps: (1) Take ginseng slices and decoct them to obtain an extract. After concentrating the extract, add ethanol for primary alcohol precipitation. Add ethanol to the supernatant after primary alcohol precipitation and let it stand for secondary alcohol precipitation. Centrifuge and collect the precipitate to obtain crude ginseng polysaccharide. Remove protein to obtain refined ginseng polysaccharide. (2) Dissolve the refined ginseng polysaccharide in distilled water and load it onto a pre-equilibrated DEAE-52 cellulose column. Perform gradient elution with sodium chloride aqueous solutions of concentrations of 0 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M and 0.40 M. Collect fractions in 12 mL / tube. Determine the sugar content in each tube of eluent using the phenol-sulfuric acid method. Plot the elution curve. Combine the characteristic elution segments according to the elution curve. Collect the elution fraction of the 5th chromatographic peak and name it GP40025. (3) Take the GP40025, dissolve it in NaCl aqueous solution to prepare a 5 mg / mL solution, load it onto a Sephadex G-75 gel chromatography column, use 0.01% NaCl aqueous solution as eluent, flow rate 1 mL / min, collect 2 mL per tube, collect 60 tubes, detect its peak shape and retention time by liquid chromatography, collect appropriate elution peaks, desalt it by Sephadex G-15 gel column chromatography, freeze dry under reduced pressure to obtain the ginseng polysaccharide GP40025-1.
2. The preparation method according to claim 1, characterized in that, The decoction conditions are as follows: the weight ratio of ginseng slices to water is 1:10; The conditions for concentrating the extract are to concentrate it to one-fifth to one-tenth of its volume.
3. The preparation method according to claim 1, characterized in that, The primary alcohol precipitation method is as follows: add 95% ethanol by volume until the ethanol concentration in the system reaches 20% (v / v), and let it stand at room temperature for 12 hours.
4. The preparation method according to claim 1, characterized in that, The conditions for the second ethanol precipitation are as follows: add 95% ethanol by volume to a final ethanol concentration of 40% (v / v) and let stand at room temperature for 12 h.
5. The preparation method according to claim 1, characterized in that, The ratio of the refined ginseng polysaccharide to distilled water is 4.0 g: 30 mL.
6. Ginseng polysaccharide GP40025-1 prepared by the preparation method according to any one of claims 1-5.
7. The ginseng polysaccharide GP40025-1 according to claim 6, characterized in that, Its structural formula is as follows: 。 8. The use of ginseng polysaccharide GP40025-1 as described in claim 6 or 7 in the preparation of products that enhance immunity.