Homogeneous polysaccharide from citrus grandis (l.) osbeck and preparation method and application thereof

Abstract

The application discloses a uniform Fructus aurantii polysaccharide and a preparation method and application thereof, and comprises the following steps: after defatting Fructus aurantii powder by petroleum ether, the Fructus aurantii powder is sequentially subjected to hot water extraction, primary alcohol precipitation, secondary alcohol precipitation, precipitation redissolution, protein removal by a Sevag method and decolorization to obtain Fructus aurantii crude polysaccharide; the Fructus aurantii crude polysaccharide is separated by anion exchange chromatography and dextran exclusion gel chromatography in series to obtain the uniform Fructus aurantii polysaccharide. The uniform Fructus aurantii polysaccharide is obtained by extraction and separation from Fructus aurantii for the first time, and the pharmacodynamic study shows that the uniform Fructus aurantii polysaccharide has the action of inhibiting hepatoma cell activity, thereby providing a new thought for developing and preparing an anti-human hepatoma cell drug; the preparation process is simple, the uniform polysaccharide can be quickly obtained, and the application is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to uniform polysaccharides of Citri Grandis Exocarpium, a preparation method and application thereof. BACKGROUND

[0002] Traditional Chinese medicine polysaccharides are an important component of polysaccharides. In recent years, with the development of cell molecular biology and glyobiology, scholars have found that traditional Chinese medicine polysaccharides have extremely wide biological activities, such as immunomodulation, antitumor, antioxidant, antiviral, anti-radiation, anti-aging, hypoglycemic, hypolipidemic, anti-inflammatory, anti-infection, regulation of intestinal flora, liver protection and analgesia. Traditional Chinese medicine polysaccharides can be prepared by various methods, but the traditional Chinese medicine polysaccharides after extraction and purification are still a complex mixed system. In order to facilitate subsequent structure characterization and biological activity research, the concept of 'uniform polysaccharides' is proposed. Uniform polysaccharides refer to polysaccharide fragments with single relative molecular mass and uniform distribution obtained by separation of total polysaccharides. Compared with total polysaccharides, uniform polysaccharides have simpler composition and more stable properties. In recent years, the preparation, structure analysis, quality evaluation and control and biological activity research of traditional Chinese medicine uniform polysaccharides are a new trend of polysaccharide research.

[0003] Citri Grandis Exocarpium is the dried pericarp of immature or nearly mature Citrus grandis 'Tomentosa' or Citrus grandis (L.) Osbeck. At present, the research on chemical components of Citri Grandis Exocarpium mainly focuses on flavonoids, volatile oils and coumarin small molecule compounds, and the research on sugar compounds, especially polysaccharides, is very little. As a fruit source of traditional Chinese medicine, it contains rich polysaccharide substances, and at present, the research on polysaccharide components of Citri Grandis Exocarpium is limited. However, polysaccharides have large molecular weight and complex chemical structure, resulting in unstable biological activity evaluation. Uniform polysaccharides are polysaccharide fragments with concentrated molecular weight distribution, small molecular weight, relatively simple chemical structure and stable chemical properties, and the preparation process is controllable, so as to facilitate the stability and reliability of the efficacy evaluation. However, there is no research report on uniform polysaccharides of Citri Grandis Exocarpium, and there is no activity research report on uniform polysaccharides of Citri Grandis Exocarpium. SUMMARY

[0004] To solve the above technical problems, the purpose of the present application is to provide a uniform polysaccharide of Citri Grandis Exocarpium, a preparation method and application thereof.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a preparation method of uniform polysaccharides of Citri Grandis Exocarpium, comprising the following steps:

[0007] (1) The Citrus grandis (L.) Osbeck powder is defatted by petroleum ether, and then sequentially subjected to hot water extraction, primary alcohol precipitation, secondary alcohol precipitation, re-dissolution of the precipitate, protein removal by the Sevag method, and decolorization to obtain crude Citrus grandis (L.) Osbeck polysaccharides;

[0008] (2) The crude Citrus grandis (L.) Osbeck polysaccharides are separated by anion exchange chromatography and dextran exclusion gel chromatography to obtain uniform Citrus grandis (L.) Osbeck polysaccharides.

[0009] Preferably, in step (1), the primary alcohol precipitation uses ethanol with a mass concentration of 75-85%, and the secondary alcohol precipitation uses ethanol with a mass concentration of 90-100%.

[0010] Preferably, in step (1), the Sevag method for protein removal uses Sevag reagent with chloroform: n-butanol = 5:1 V / V; after protein removal, 30% hydrogen peroxide solution is added to reach a volume of 15%, and the solution is placed in a 60°C water bath for decolorization.

[0011] Preferably, in step (2), the anion exchange chromatography uses a DEAE-52 cellulose chromatography column, the crude Citrus grandis (L.) Osbeck polysaccharides are loaded, and then sequentially eluted with distilled water, 0.1, 0.2, and 0.4 mol / L NaCl solutions; the phenol-sulfuric acid method is used for real-time monitoring, and the eluate after elution with distilled water and 0.4 mol / L NaCl solution is collected to obtain CGEPP-H2O and CGEPP-0.4M polysaccharides, respectively.

[0012] Preferably, in step (2), the dextran gel chromatography uses a SephacryL-S-300HR propylene dextran gel chromatography column, the CGEPP-0.4M polysaccharides are loaded, and then eluted with distilled water; the eluate is determined for ultraviolet absorbance value by the phenol-sulfuric acid method, and an elution curve is drawn with tube number as the horizontal coordinate and absorbance value as the vertical coordinate; and according to the elution curve, the elution peak position is combined, and then concentrated under reduced pressure and dried to obtain uniform Citrus grandis (L.) Osbeck polysaccharides CGEHP-1 and uniform Citrus grandis (L.) Osbeck polysaccharides CGEHP-2.

[0013] The SephacryL-S-300HR propylene dextran gel chromatography column has excellent resolution and flow characteristics, high physical and chemical stability, and is easy to operate.

[0014] In a second aspect, the present application provides a uniform Citrus grandis (L.) Osbeck polysaccharide prepared by the above preparation method.

[0015] Analysis and identification show that the uniform Citrus grandis (L.) Osbeck polysaccharide CGEHP-1 has a relative molecular weight of 11310.56 Da and is composed of rhamnose, arabinose, mannose, and glucose; and the uniform Citrus grandis (L.) Osbeck polysaccharide CGEHP-2 has a relative molecular weight of 8112.34 Da and is composed of single mannose.

[0016] In a third aspect, the present application provides the use of the uniform polysaccharide in Fructus aurantii in the preparation of a medicine for resisting human hepatoma cells.

[0017] The medicine comprises the uniform polysaccharide in Fructus aurantii and pharmaceutically acceptable excipients. The medicine can be prepared into a suitable dosage form by a conventional method in the art, and preferably, the dosage form of the medicine is a capsule, a tablet, a pill, a granule or an oral liquid.

[0018] Compared with the prior art, the present application has the following excellent effects:

[0019] (1) The uniform polysaccharide in Fructus aurantii is extracted and separated from Fructus aurantii for the first time, and the pharmacodynamic study shows that the uniform polysaccharide in Fructus aurantii has the effect of inhibiting hepatoma cells, thereby providing a new idea for developing and preparing a medicine for resisting human hepatoma cells.

[0020] (2) The preparation process of the present application is simple, and the uniform polysaccharide can be quickly obtained, which is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Elution curves for column chromatography separation, A is the elution curve of the crude polysaccharide in Fructus aurantii for anion exchange column chromatography, B is the elution curve of the crude polysaccharide in Fructus aurantii for CGEPP-0.4M dextran gel exclusion chromatography, and C is the elution curve of the crude polysaccharide in Fructus aurantii for CGEPP-H2O dextran gel exclusion chromatography;

[0022] Figure 2 UPLC-SEC-ELSD chromatogram of a series of dextran reference solution;

[0023] Figure 3 UPLC-SEC-ELSD chromatogram of different molecular weight fragments of the polysaccharide in Fructus aurantii;

[0024] Figure 4 TIC graph of a monosaccharide inositol mixed standard;

[0025] Figure 5 TIC graph of monosaccharide composition analysis in the uniform polysaccharide CGEHP-1 and CGEHP-2 in Fructus aurantii, A is the uniform polysaccharide CGEHP-1 in Fructus aurantii, and B is the uniform polysaccharide CGEHP-2 in Fructus aurantii;

[0026] ˉ

[0027] Figure 6 HepG2 cell viability graph under the action of a series of concentrations of the uniform polysaccharide CGEHP-1 in Fructus aurantii (n=3, x±s, *p<0.05, **p<0.01 compared with the 0 μg / mL group);

[0028] ˉ

[0029] Figure 7Figure 1 shows the cell viability of HepG2 cells under the action of different concentrations of the series of uniform polysaccharides CGEHP-2 (n = 3, x ± s, *p < 0.05, **p < 0.01 compared with the 0 μg / mL group). DETAILED DESCRIPTION

[0030] The application will be further described below by means of specific embodiments, and the following examples are specific embodiments of the application, but the embodiments of the application are not limited by the following examples.

[0031] Example 1:

[0032] (1) Pretreatment: Take the medicinal material of Fructus Aurantii, clean and select, and then crush, soak in petroleum ether overnight, defat, and evaporate the solvent;

[0033] (2) Extraction: Add 14-16 times the amount of water to the pretreated medicinal material of Fructus Aurantii, soak and then heat to extract, extract 1-2 times, each time for 1 h, filter, combine the filtrate, and concentrate under reduced pressure to obtain the water extract of Fructus Aurantii;

[0034] (3) First alcohol precipitation: Add anhydrous ethanol to the water extract of Fructus Aurantii to make the alcohol content reach 80%, stand at room temperature for 1 day, filter under reduced pressure to take the precipitate, and dry at 60°C under vacuum to obtain the crude polysaccharide extract of Fructus Aurantii;

[0035] (4) Second alcohol precipitation: Take the crude polysaccharide extract of Fructus Aurantii, redissolve with appropriate distilled water, add anhydrous ethanol to make the alcohol content reach 95%, stand overnight in a 4°C environment, filter under reduced pressure to take the precipitate, wash repeatedly with 95% ethanol for 3 times, and dry under vacuum at 65°C to obtain the crude polysaccharide mixture of Fructus Aurantii;

[0036] (5) Purification: Prepare Sevag reagent according to the mixture of chloroform: n-butanol = 5:1 (V / V), mix the crude polysaccharide mixture solution with the Sevag reagent according to the ratio of 5:1, and then place in a separatory funnel, shake fully for 20 min, recover the crude polysaccharide extract, repeat multiple times until there is no protein precipitate at the interface between the polysaccharide extract and the Sevag reagent, then add 30% hydrogen peroxide solution, stand in a 60°C water bath for 6 h, centrifuge to take the supernatant, vacuum concentrate, and freeze-dry to obtain the crude polysaccharide CGECP of Fructus Aurantii;

[0037] (6) Initial separation: A DEAE-52 cellulose chromatography column with XK 25mm×200mm was used. After activation, the DEAE-52 cellulose packing material was wet-packed and sealed with absorbent cotton. The column was equilibrated with 4 BV of distilled water. The crude polysaccharide CGECP of Citrus reticulata was fully dissolved in distilled water to prepare a solution. Each time, 10 mL of the CGECP solution was filtered through a 0.45 μm aqueous filter membrane and loaded onto the column. Gradient elution was performed sequentially with distilled water, 0.1, 0.2, and 0.4 mol / L NaCl solutions. During the elution process, a constant flow pump was used to control the flow rate. The eluent was collected immediately after loading using graduated centrifuge tubes, collecting 3 mL per tube. 100 tubes of each concentration gradient eluent were collected. The absorbance of each tube of eluent was measured sequentially using the phenol-sulfuric acid method according to the elution concentration. The elution curve was plotted with the number of tubes as the x-axis and the absorbance value as the y-axis (e.g., ...). Figure 1 (As shown in A); collect the eluent from the peak of the elution curve, filter the high-response component (distilled water component and 0.4 mol / L NaCl component eluent) through a 0.45 μm microporous membrane, and concentrate it to the same amount as the total amount loaded, to obtain CGEPP-H2O and CGEPP-0.4M polysaccharide, respectively.

[0038] (7) Secondary separation: Further separation was performed using an propylene dextran gel chromatography column with parameters of XK20mm×90cm. GE Healthcare SephacryL-S-300HR packing material was selected. After pretreatment, the column was wet-packed and equilibrated with 2 BV distilled water before loading. 10 mL of CGEPP-H2O and CGEPP-0.4M polysaccharide were loaded each time, with distilled water as the eluent. Collection began after loading using graduated centrifuge tubes and a constant flow pump to control the elution rate. 3 mL of eluent was collected from each tube, for a total of 100 tubes. The absorbance of the eluent was measured using the phenol-sulfuric acid method. An elution curve was plotted with the tube number on the x-axis and the absorbance value on the y-axis (e.g., ...). Figure 1 (As shown in B and C); and based on the elution curves, the elution peaks were merged, and after vacuum concentration and freeze drying, the homogeneous polysaccharide CGEHP-1, homogeneous polysaccharide CGEHP-2, and heterogeneous polysaccharide component CGEHP-W of Citrus reticulata were prepared.

[0039] The prepared homogeneous polysaccharide (CGEHP) was identified, and the specific process is as follows:

[0040] Column: TOSOH CORPORATION TSKgel GMPW XL (Column Size: 7.8mm ID×30cm, Particle Size: 13μm), column temperature 30℃;

[0041] LC conditions: mobile phase was ultrapure water through 0.22 μm, flow rate was 0.3 mL / min, single injection was 20 μL, signal collection time was 45 min;

[0042] ELSD conditions: Tube Tem (drift tube temperature) was 100 ℃, Gas Flow (carrier gas flow rate) was high-purity nitrogen, 2.5 L / min, baffle closed mode.

[0043] Take dextran series standard T3 (Mw=3.03×10 3 ), T10 (Mw=1.26×10 4 ), T70 (Mw=6.33×10 4 ), T100 (Mw=1.26×10 5 ), T300 (Mw=3.02×10 5 ) and T500 (Mw=5.56×10 5 ) powders, add appropriate amount of distilled water to prepare a control solution with a concentration of about 1.0 mg / mL, filter through a 0.22 μm water-based microporous filter;

[0044] Preparation of test solution: take prepared Citrus grandis (L.) Osbeck uniform polysaccharide CGEHP-1, Citrus grandis (L.) Osbeck uniform polysaccharide CGEHP-2, Citrus grandis (L.) Osbeck polysaccharide CGEHP-W and Citrus grandis (L.) Osbeck crude polysaccharide CGECP sample powder, add appropriate amount of distilled water to completely dissolve, prepare a test solution with a mass concentration of about 1.0 mg / mL, filter through a 0.22 μm water-based microporous filter. The series dextran control solution and the test solution are detected according to the conditions. With the retention time tR as the abscissa and the logarithmic value LgMw of the weight average molecular weight of the series dextran control as the ordinate, the standard curve is obtained by regression analysis. The Citrus grandis (L.) Osbeck uniform polysaccharide CGEHP-1, Citrus grandis (L.) Osbeck uniform polysaccharide CGEHP-2, Citrus grandis (L.) Osbeck polysaccharide CGEHP-W and Citrus grandis (L.) Osbeck crude polysaccharide CGECP test solution are detected for uniformity and molecular weight according to the same method.

[0045] Figure 2 The regression curve of the peak time of the series dextran control-Mw logarithmic value is shown; the curve equation is y=-0.3849x+15.222, R 2 =0.9993, indicating that the linear relationship between the obtained peak time and the Mw logarithmic value is good, which can be used to calculate the relative molecular weight of the polysaccharide sample solution. It is calculated that the relative molecular weight of Citrus grandis (L.) Osbeck uniform polysaccharide CGEHP-1 is 11310.56 Da, and the relative molecular weight of CGEHP-2 is 8112.34 Da.

[0046] Figure 3The UPLC-SEC-ELSD chromatogram of the different molecular weight fragments of the polysaccharides from Citrus grandis (L.) Osbeck is shown in Figure 1, where A is the chromatogram of the crude polysaccharides CGECP from Citrus grandis (L.) Osbeck, B is the chromatogram of the polysaccharides CGEHP-W from Citrus grandis (L.) Osbeck, C is the chromatogram of the uniform polysaccharides CGEHP-1 from Citrus grandis (L.) Osbeck, and D is the chromatogram of the uniform polysaccharides CGEHP-2 from Citrus grandis (L.) Osbeck. According to the determination criteria, when the size exclusion chromatography peak is a single peak, it is determined to be a single component with uniform molecular weight distribution.

[0047] According to the determination results, the crude polysaccharides CGECP from Citrus grandis (L.) Osbeck are non-uniform components, the polysaccharides CGEHP-W are non-uniform components, and the uniform polysaccharides CGEHP-1 and CGEHP-2 are uniform components. Figure 3

[0048] The monosaccharide composition of the prepared uniform polysaccharides (CGEHP) from Citrus grandis (L.) Osbeck was identified, and the specific process is as follows:

[0049] The uniform polysaccharides CGEHP-1 and CGEHP-2 from Citrus grandis (L.) Osbeck were subjected to acid hydrolysis and sugar nitrile acetylation treatment, and then the monosaccharide composition was analyzed by gas chromatography-mass spectrometry (GC-MS). Agilent GC-MS, model 5977A-7890B, Angilent Technologies was used.

[0050] 1. Acid hydrolysis of polysaccharide samples

[0051] About 10.0 mg of the uniform polysaccharides CGEHP-1 and CGEHP-2 from Citrus grandis (L.) Osbeck was weighed into a 5 mL ampoule, 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution was added, and the ampoule was sealed by a spray gun at high temperature and placed in a water bath at 110°C for 2 h. After the reaction was completed, the ampoule was cooled to room temperature, 2 mL of analytical grade methanol was added, and the ampoule was dried at 65°C under vacuum. The above steps were repeated four times to remove the residual TFA. The sample was placed in a vacuum environment for standby after the last treatment, and the uniform polysaccharide hydrolysate from Citrus grandis (L.) Osbeck was obtained.

[0052] 2. Preparation of monosaccharide and inositol sugar nitrile acetylation standard solution

[0053] ​The standard L-rhamnose, L-arabinose, D-(+)-xylose, D-mannose, D(+)-glucose, D-galactose and L(-) fucose, inositol about 2.0 mg were weighed into 15 mL centrifuge tubes, and 1.0 mL of 50 mg / mL hydroxylamine hydrochloride-pyridine solution was added, and ultrasonic was used to completely dissolve to obtain monosaccharide single standard, inositol single standard pretreatment solution. 100 μL of each group of single standard pretreatment solution was taken and mixed uniformly in a 15 mL centrifuge tube to obtain a mixed standard pretreatment solution. All centrifuge tubes were placed in a 90°C constant temperature water bath oscillator for reaction for 40 min, and the centrifuge tube opening was covered with sealing film to prevent moisture from entering. After the reaction was completed, it was cooled to room temperature, and an equal volume of acetic anhydride was added to each group of hydroxylamine hydrochloride-pyridine, sealed, ultrasonically mixed, and placed in a 90°C constant temperature water bath oscillator for reaction for 40 min. After standing at room temperature, high-speed centrifugation was performed at 4000 r / min for 10 min, the upper layer solution was taken, filtered with a 0.22 μm organic filter membrane, and placed in a liquid phase bottle to obtain the monosaccharide and inositol sugar nitrile acetylated derivative control solution.

[0054] 3. Preparation of Citrus grandis (L.) Osbeck uniform polysaccharide hydrolysate glycan acetylated derivative

[0055] The Citrus grandis (L.) Osbeck uniform polysaccharide hydrolysate was also prepared according to the above-mentioned “glycan acetylated control solution preparation” method to obtain the Citrus grandis (L.) Osbeck uniform polysaccharide glycan acetylated derivative test solution.

[0056] 4. GC-MS analysis conditions

[0057] GC-MS conditions: the chromatographic column was an HP-5ms Ultra Inert capillary column; the injection port temperature was 230°C; the split ratio was 10:1; the carrier gas was high-purity He; the injection amount was 1.5 μL; the programmed temperature was: the initial temperature was 70°C, maintained for 8 min, increased to 160°C at a rate of 10°C / min, maintained for 20 min, increased to 170°C at a rate of 10°C / min, maintained for 13 min, and increased to 220°C at a rate of 10°C / min, maintained for 3 min. MS conditions: the detector temperature was 230°C, the fragment ions scanned in the Scan mode were m / z 50-550, and the ion energy was 70 eV voltage. The prepared monosaccharide single standard derivative, monosaccharide mixed standard derivative and Citrus grandis (L.) Osbeck uniform polysaccharide derivative were used for qualitative analysis by GC-MS.

[0058] As Figures 4-5As shown, through comparison analysis, it is known that the uniform polysaccharide CGEHP-1 component of Cheniqi Hong is composed of rhamnose, arabinose, mannose and glucose, and the uniform polysaccharide CGEHP-2 component of Cheniqi Hong is composed of single mannose.

[0059] Hepatoma cell toxicity inhibition verification experiment of uniform polysaccharide of Cheniqi Hong:

[0060] Take the cells in the logarithmic growth phase for the experiment. After trypsin digestion, the logarithmic growth phase HepG2 cells are inoculated into a 96-well plate, 100 μL of cell suspension per well, and the cell concentration is adjusted to 1.0×105 cells per well after counting by the cell automatic technology instrument. Place in a carbon dioxide incubator for 12 h, conditions 37℃, 5% CO2 concentration. After the cells adhere, discard the upper culture medium, wash with PBS buffer and aspirate the residual liquid. Use DMEM blank culture medium to prepare solutions with concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, 400, 800 and 1000 μg / mL. Observe the growth status of the cells under an inverted microscope, and when the number of cells accounts for 70%-80% of the observation field, add different concentrations of uniform polysaccharide CGEHP-1 and CGEHP-2 in each well and mark well, set 6 replicate wells for each group, and take out after 24 h of culture in the original culture environment. Pour the upper culture medium, wash with PBS and aspirate the residual reagent, add 100 μL of DMEM blank culture medium and 10 μL of CCK-8 reagent to each well, transfer to the incubator for 4 h, then take out, and measure the absorbance value at 450 nm using an enzyme marker. Take the blank group cell activity as 100%, and calculate the cell activity value of each group. Cell viability = A drug group / A blank group*100%. Use SPSS 26.0 software to analyze the data. The results are shown in Figures 1-2 and Figures 6-7 .

[0061] Table 1 Effect of different concentrations of CGEHP-1 on HepG2 cell activity (n = 3, )

[0062]

[0063]

[0064] Table 2 Effect of different concentrations of CGEHP-2 on HepG2 cell activity (n = 3, )

[0065]

[0066] From the above results, it can be seen that: the uniform polysaccharide CGEHP-1 and the uniform polysaccharide CGEHP-2 prepared by anion exchange column chromatography and gel exclusion chromatography from the crude polysaccharide of Citrus grandis (L.) Osbeck ex Tanaka have the activity of inhibiting HepG2 cells, and the inhibition effect of the uniform polysaccharide CGEHP-2 shows obvious dose dependence. The whole process is clear, the method for judging the uniformity of the product is feasible and easy to obtain, and the cell toxicity is clear. It can provide experimental data for the material basis research of the polysaccharide components of Citrus grandis (L.) Osbeck ex Tanaka, and can be popularized.

Claims

1. The use of uniform polysaccharides from Citrus grandis (L.) Osbeck in the preparation of a medicine for resisting human hepatoma cells; the relative molecular weight of the uniform polysaccharide CGEHP-1 from Citrus grandis (L.) Osbeck is 11310.56 Da, and it is composed of rhamnose, arabinose, mannose and glucose; the relative molecular weight of the uniform polysaccharide CGEHP-2 from Citrus grandis (L.) Osbeck is 8112.34 Da, and it is composed of single mannose; The preparation method of the uniform polysaccharides from Citrus grandis (L.) Osbeck comprises the following steps: (1) after defatting the powder of Citrus grandis (L.) Osbeck with petroleum ether, the powder is sequentially subjected to hot water extraction, primary alcohol precipitation, secondary alcohol precipitation, precipitation redissolution, protein removal by the Sevag method and decolorization to obtain crude polysaccharides from Citrus grandis (L.) Osbeck; (2) the crude polysaccharides from Citrus grandis (L.) Osbeck are separated by anion exchange chromatography and dextran exclusion gel chromatography in series to obtain uniform polysaccharides from Citrus grandis (L.) Osbeck; The anion exchange chromatography adopts a DEAE-52 cellulose chromatography column, and after the crude polysaccharides from Citrus grandis (L.) Osbeck are loaded, the column is sequentially eluted with distilled water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.4 mol / L NaCl solution, and the eluate after elution with distilled water and 0.4 mol / L NaCl solution is collected, concentrated and then CGEPP-H2O and CGEPP-0.4M polysaccharides are obtained, respectively; The dextran exclusion gel chromatography adopts a SephacryL-S-300HR propylene dextran gel chromatography column, and after the CGEPP-0.4M polysaccharides are loaded, the column is eluted with distilled water, and the eluate is determined for ultraviolet absorbance value by the phenol-sulfuric acid method, and an elution curve is drawn with the tube number as the horizontal coordinate and the absorbance value as the vertical coordinate; and according to the elution curve, the elution peak parts are combined, concentrated under reduced pressure and dried to obtain the uniform polysaccharides CGEHP-1 and CGEHP-2 from Citrus grandis (L.) Osbeck.

2. Use according to claim 1, characterized in that, In step (1), the primary alcohol precipitation adopts ethanol with a mass concentration of 75-85 %, and the secondary alcohol precipitation adopts ethanol with a mass concentration of 90-100 %.

3. Use according to claim 1, characterized in that, In step (1), the Sevag reagent used for protein removal by the Sevag method is chloroform:n-butanol=5:1 V / V; after protein removal, a hydrogen peroxide solution with a mass concentration of 30 % is added to make the volume reach 15 %, and the solution is placed in a water bath at 60 ℃ for decolorization.

4. Use according to claim 1, characterized in that, The medicine comprises the uniform polysaccharides from Citrus grandis (L.) Osbeck and pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that, The dosage form of the medicine is a capsule, a tablet, a pill, a granule or an oral liquid.

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