Qu fructus aurantii polysaccharide as well as preparation method and application thereof

Abstract

The invention provides Qu fructus aurantii polysaccharide as well as a preparation method and application thereof, and relates to the technical field of Qu fructus aurantii polysaccharide. The Qu fructus aurantii polysaccharide is prepared from galacturonic acid, arabinose, galactose, rhamnose, mannose, fucose, glucuronic acid, glucose and xylose. The Qu fructus aurantii polysaccharide is obtained through water extraction, alcohol precipitation, deproteinization and dialysis. An in-vitro macrophage experiment shows that the Qu fructus aurantii polysaccharide has the effects of enhancing the phagocytic activity of macrophages, promoting the macrophages to release inflammatory factors IL-1beta, IL-10, IL-6, TNF-alpha and NO and promoting the macrophages to release ROS, and the Qu fructus aurantii polysaccharide has the immunopotentiation effect.

Description

Technical Field

[0001] The invention belongs to the technical field of Quzhizhi polysaccharide, and specifically relates to Quzhizhi polysaccharide and a preparation method and application thereof. Background Art

[0002] Quzhike (green pomelo) is the dried immature fruit of the Rutaceae plant Citrus changshan-huyou YBChang. It has the effects of regulating qi, relieving fullness, relieving stagnation and relieving bloating. It has a long history of being used as a folk medicine, and is often used to treat respiratory diseases and digestive system diseases.

[0003] Polysaccharides are a class of natural high molecular polymers formed by aldose or ketose connected by glycosidic bonds. They are widely present in organisms and are important biological macromolecules in organisms. Plant polysaccharides are polysaccharides formed by the condensation of more than 10 monosaccharide molecules in plants. Modern research has shown that many plant polysaccharides have a wide range of biological activities, including immunomodulation, anti-tumor, hypoglycemic, hypolipidemic, anti-radiation, antibacterial and antiviral, and liver protection. Therefore, plant polysaccharides play an important role in the treatment of various diseases and health care. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a polysaccharide from Quzhizhi and a preparation method thereof, wherein the polysaccharide from Quzhizhi has the function of enhancing the phagocytic activity of macrophages and promoting the release of inflammatory factors by macrophages.

[0005] Another object of the present invention is to provide an application of Chitosan polysaccharide in the preparation of medicines or health foods for enhancing immunity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a chitosan polysaccharide. Calculated by mole percentage, the monosaccharide composition of the chitosan polysaccharide is 60.2-62.2% of galacturonic acid, 13.6-15.6% of arabinose, 19.0-21.0% of galactose, 2.5-3.5% of rhamnose, 0.3-0.5% of mannose, 0.2-0.4% of fucose, 0.2-0.4% of glucuronic acid, 0.05-0.15% of glucose and 0.05-0.15% of xylose.

[0008] Preferably, taking dextran as the standard reference, the total polysaccharide content of the Quzhizhi polysaccharide is 90.96-96.96%; taking galacturonic acid as the standard reference, the uronic acid content of the Quzhizhi polysaccharide is 60.49-66.49%.

[0009] The present invention also provides a method for preparing the chitosan polysaccharide, comprising the following steps:

[0010] Extracting the Fructus Aurantii Immaturus with water to obtain a Fructus Aurantii Immaturus water extract; concentrating the Fructus Aurantii Immaturus water extract under reduced pressure and subjecting it to alcohol precipitation to obtain a precipitate; re-dissolving the precipitate with water, performing chromatography, elution, concentrating under reduced pressure, and freeze-drying to obtain a crude polysaccharide of Fructus Aurantii Immaturus;

[0011] The crude polysaccharide of Quzhizi is enzymatically hydrolyzed to obtain an enzymatic hydrolyzate; Sevage solution is added to the enzymatic hydrolyzate for deproteinization; after the deproteinization, the water layer is placed in a dialysis bag for dialysis, and freeze-dried to obtain the polysaccharide of Quzhizi.

[0012] Preferably, during water extraction, the solid-liquid ratio of Quyi Fructus and water is 1g:8mL to 1g:12mL.

[0013] Preferably, the Fructus Aurantii Immaturus water extract is concentrated under reduced pressure to 5% to 10% of the original volume.

[0014] Preferably, the chromatographic adsorption material is AB-8 macroporous adsorption resin.

[0015] Preferably, the enzyme for enzymolysis is papain; and the conditions for enzymolysis are enzymolysis in a water bath at 55-65° C. for 0.5-2 h.

[0016] Preferably, the specification of the dialysis bag is a dialysis bag with a molecular weight cut-off of 3500Da.

[0017] The present invention also provides an application of the chitosan polysaccharide in preparing medicines or health-care foods for enhancing immunity.

[0018] The present invention also provides a pharmaceutical composition, which comprises the Chitosan polysaccharide and pharmaceutically acceptable excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention obtains Quzhi chitosan polysaccharide by water extraction, alcohol precipitation, deproteinization and dialysis. The present invention finds through in vitro macrophage experiments that Quzhi chitosan polysaccharide has the ability to enhance the phagocytic activity of macrophages, promote macrophages to release inflammatory factors IL-1β, IL-10, IL-6, TNF-α and NO, and promote macrophages to release ROS, indicating that the Quzhi chitosan polysaccharide prepared by the present invention has an immunoenhancing effect. The Quzhi chitosan polysaccharide of the present invention has an immunoenhancing effect, and has broad application prospects in the preparation of drugs or health foods that enhance immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a high-performance gel permeation chromatogram of the chitosan polysaccharide of the present invention;

[0022] Figure 2is the ion chromatogram of the monosaccharide standard;

[0023] Figure 3 is the ion chromatogram of monosaccharides in the chitosan polysaccharide of the present invention;

[0024] Figure 4 The polysaccharide of the present invention enhances the phagocytic activity of macrophages. In the figure, A is the OD value of macrophage activity under treatment with different concentrations of polysaccharide of the present invention, and B is the change of macrophage phagocytic ability under different treatment groups. Compared with the Ctrl group, ***P<0.001;

[0025] Figure 5 The chitosan polysaccharide of the present invention promotes the release of inflammatory factors in macrophages. Compared with the Ctrl group, ***P<0.001;

[0026] Figure 6 The chitosan polysaccharide of the present invention promotes the release of ROS in macrophages. Compared with the Ctrl group, ***P<0.001. DETAILED DESCRIPTION

[0027] The present invention provides a polysaccharide of Quzhizhi. In terms of mole percentage, the monosaccharide composition of the polysaccharide of Quzhizhi is preferably 60.2-62.2% of galacturonic acid, 13.6-15.6% of arabinose, 19.0-21.0% of galactose, 2.5-3.5% of rhamnose, 0.3-0.5% of mannose, 0.2-0.4% of fucose, 0.2-0.4% of glucuronic acid, 0.05-0.15% of glucose and 0.05-0.15% of xylose; in terms of mole percentage, the monosaccharide composition of the polysaccharide of Quzhizhi is more preferably 61.2% of galacturonic acid, 14.6% of arabinose, 20.0% of galactose, 3.0% of rhamnose, 0.4% of mannose, 0.3% of fucose, 0.2% of glucuronic acid, 0.1% of glucose and 0.1% of xylose. The present invention preferably adopts ion chromatography to determine the monosaccharide composition of Chitosan polysaccharide.

[0028] The present invention preferably uses glucan as a standard control and adopts the sulfuric acid phenol method to determine the content of Quzhizhi polysaccharide. After determination, the total polysaccharide content of the Quzhizhi polysaccharide is preferably 90.96-96.96%, and more preferably 93.96%. The present invention preferably uses galacturonic acid as a standard control and adopts the sulfate carbazole method to determine the uronic acid content in Quzhizhi polysaccharide. After determination, the uronic acid content of the Quzhizhi polysaccharide is preferably 60.49-66.49%, and more preferably 63.49%.

[0029] The present invention also provides a method for preparing the chitosan polysaccharide, comprising the following steps:

[0030] Extracting the Fructus Aurantii Immaturus with water to obtain a Fructus Aurantii Immaturus water extract; concentrating the Fructus Aurantii Immaturus water extract under reduced pressure and subjecting it to alcohol precipitation to obtain a precipitate; re-dissolving the precipitate with water, performing chromatography, elution, concentrating under reduced pressure, and freeze-drying to obtain a crude polysaccharide of Fructus Aurantii Immaturus;

[0031] The crude polysaccharide of Quzhizi is enzymatically hydrolyzed to obtain an enzymatic hydrolyzate; Sevage solution is added to the enzymatic hydrolyzate for deproteinization; after the deproteinization, the water layer is placed in a dialysis bag for dialysis, and freeze-dried to obtain the polysaccharide of Quzhizi.

[0032] In the present invention, the Quzhike raw material is preferably Quzhike medicinal material or Quzhike decoction pieces, and the Quzhike raw material can be purchased through conventional channels. In the present invention, the Quzhike raw material is added with water for water extraction, and the number of water extractions is preferably 2 to 3 times. After water extraction, the extracts are combined to obtain the Quzhike water extract. In the present invention, during water extraction, the material-liquid ratio of Quzhike to water is preferably 1g:8mL to 1g:12mL, and more preferably 1g:10mL.

[0033] In the present invention, the obtained Quzhi Fructus water extract is concentrated under reduced pressure, preferably the Quzhi Fructus water extract is concentrated under reduced pressure to 5% to 10% of the original volume, more preferably 6% to 8%. After reduced pressure concentration, preferably 70 to 90% ethanol concentration is used for alcohol precipitation, the temperature of the alcohol precipitation is preferably 0 to 7°C, more preferably the ethanol concentration is 80%, and the alcohol precipitation temperature is 2 to 4°C; after alcohol precipitation, the precipitate is filtered to obtain the precipitate, the precipitate is suspended with 80% ethanol, centrifuged, and repeated 3 to 4 times to obtain the final precipitate. The final precipitate is redissolved in water, chromatographed by AB-8 macroporous adsorption resin column, eluted with 3 to 7BV water, and the water eluate is concentrated under reduced pressure to a small volume, freeze-dried, and the crude Quzhi Fructus polysaccharide is obtained. In the present invention, the final precipitate loading amount and resin mass ratio are preferably 1:3 to 1:5, more preferably 1:4.

[0034] In the present invention, the crude polysaccharide of Quzhizi is dissolved in water, and then papain is added, and enzymolysis is carried out in a water bath at 55-65°C for 0.5-2h, and then cooled; Sevage solution is added and shaken, the chloroform layer is discarded, and deproteinization is repeated 4-6 times, and the water layer is dialyzed in a dialysis bag, and the solution in the dialysis bag is freeze-dried to obtain Quzhizi polysaccharide. In the present invention, the enzymolysis temperature is more preferably 60°C water bath for 1h. In the present invention, the Sevage solution is preferably prepared by mixing chloroform and n-butanol in a mass ratio of 4:1. In the present invention, as an operative method, the crude polysaccharide of Quzhizi is dissolved in water without enzymolysis, and subsequent deproteinization and chromatography are directly carried out. In the present invention, the freeze-drying conditions can be conventionally adjusted according to actual needs; the freeze-drying conditions of the present invention are preferably a freezing temperature of less than -40°C and a freezing pressure of less than 0.12mBar.

[0035] In the present invention, the specification of the dialysis bag is preferably a dialysis bag with a molecular weight cut-off of 3500Da.

[0036] The present invention also provides an application of the chitosan polysaccharide in preparing medicines or health-care foods for enhancing immunity.

[0037] The present invention also provides a pharmaceutical composition, which comprises the chitosan polysaccharide and pharmaceutically acceptable excipients. In the present invention, the pharmaceutically acceptable excipients are not particularly limited and can be conventionally selected according to actual needs. The excipients include one or more of carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and spices. In the present invention, the excipients can be specifically selected from lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil. One or more.

[0038] In the present invention, the dosage form of the drug is not particularly limited and can be conventionally selected according to actual needs. The dosage form of the drug of the present invention includes but is not limited to one or more of injection, powder injection, granule, capsule, tablet, powder, oral solution, suspension and emulsion.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0041] Example 1

[0042] A method for preparing chitosan polysaccharide, the specific steps are as follows:

[0043] 1000g of the original medicinal material of Quzhi Fructus was soaked in water, and 10 times of water was added to heat and reflux for extraction for 1.5 hours, for a total of 3 times. The three extracts were combined and concentrated under reduced pressure to 10% of the original volume, and ethanol was added to make the final concentration of 80% ethanol. The alcohol was precipitated and refrigerated overnight. Filter, suspend the precipitate with 80% ethanol, centrifuge at 4000r / min, repeat 3 times, and obtain 209.2g of precipitate. The precipitate was dissolved in hot water, chromatographed on an AB-8 (840g) macroporous adsorption resin column, eluted with 5BV water, and the water eluate was concentrated under reduced pressure to a small volume, freeze-dried, and 62.1g of Quzhi Fructus crude polysaccharide was obtained.

[0044] Take 5.0g of crude polysaccharide sample of Quzhizi, add 160mL of double distilled water, and dissolve it by ultrasonic heating. Then weigh 1.0g of papain, add it to the polysaccharide solution, shake it gently, make it swell naturally, and enzymolyze it in a water bath at 60℃ for 1h. After cooling, add Sevage solution (chloroform: n-butanol = 4:1) and shake it. Discard the chloroform layer, repeat the deproteinization 5 times, and dialyze the water layer in a dialysis bag (dialysis molecular weight 3500Da). The solution in the dialysis bag is freeze-dried to obtain 2.7g of Quzhizi polysaccharide CSHYP.

[0045] Example 2

[0046] In this example, the molecular weight of the chitosan polysaccharide CSHYP prepared in Example 1 was measured by high performance gel permeation chromatography, and the steps were as follows:

[0047] Preparation of standard solution: Accurately weigh 5 mg of each standard (dextran), dissolve in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, and transfer the sample to a 1.8 mL injection vial.

[0048] Sample solution preparation: accurately weigh 5 mg of sample, dissolve in 1 mL of mobile phase solution to prepare 5 mg / ml solution, vortex to dissolve, centrifuge at 12000 rpm for 10 min. Pipette the supernatant, filter with a 0.22 μm water microporous filter membrane, and then transfer the sample to a 1.8 mL injection vial.

[0049] Thermo UltiMate 3000 high performance liquid chromatograph, chromatographic column: BRT105-103-101 series gel column (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.7 ml / min; column temperature: 40°C; injection volume: 25 μL; analysis time: 70 min. Detector: RI-20A differential detector.

[0050] The standard solution was analyzed according to the above chromatographic method to obtain the retention time, and the standard curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), and lgMn-RT (Mn number average molecular weight) were drawn to obtain the molecular weight calculation formula. The sample solution was analyzed in the same way to obtain the chromatogram and retention time. The retention time was substituted into the formula to obtain the molecular weight (Mp, Mw, Mw). The molecular weight results of Quzhizhi polysaccharide are shown in Table 1. The high-performance gel permeation chromatogram of Quzhizhi polysaccharide is shown in Figure 1 .

[0051] Table 1 Molecular weight of chitosan polysaccharide

[0052] RT(min) lgM lm w lgMn Mp M Mn Relative peak area (%) 25.008 6.1 6.1 6.1 1255521 1289470 1257236 1.991 35.818 4.6 4.6 4.6 44145 43568 43226 96.991 44.818 3.4 3.4 3.4 2719 2596 2613 1.017

[0053] Note: The unit of molecular weight is Da

[0054] Determination of Monosaccharide Composition of Chitosan Polysaccharide from Quzhou Citrus aurantium by Ion Chromatography

[0055] Acid hydrolysis: Accurately weigh 5 mg of sample and place it in an ampoule, add 2 mL of 3M trifluoroacetic acid, and hydrolyze at 120°C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry with nitrogen, add 5 mL of water and vortex to mix, pipette 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant for ion chromatography analysis.

[0056] ThermoFisher ICS5000 ion chromatograph, column: Dionex Carbopac TM PA20 (3mm×150mm); mobile phase: A: H2O; B: 250mMNaOH; C: 500mMNaOH&50mM NaAc; elution gradient A:B:C (V / V / V): 0min 98:2:0, 23min 98:2:0, 23.1min 80:20:0, 33min 80:20:0, 33.1min 80:0:20, 46min80:0:20, 46.1min 20:0:80, 66min20:0:80, 66.1min 98:2:0, 80min 98:2:0. Flow rate: 0.3mL / min; injection volume: 25μL; column temperature: 30℃; electrical detector: chemical detector.

[0057] The monosaccharide standard solution was treated in the same manner as above. Ion chromatogram of monosaccharide standard ( Figure 2 ) and monosaccharide ion chromatograms of polysaccharides from Citrus aurantium ( Figure 3 ). The molar percentage was calculated based on the molar mass of monosaccharides. The results are shown in Table 2.

[0058] Table 2 Monosaccharide composition of Chitosan polysaccharide

[0059] Monosaccharides Retention time (RT) Peak area Mole percentage (%) Fucose 5.992 0.186 0.3 Rhamnose 12 1.349 3.0 Arabinose 12.334 15.844 14.6 Galactose 15.425 17.57 20.0 glucose 17.684 0.241 0.1 Xylose 20.675 0.183 0.1 Mannose 21.675 0.438 0.4 Galacturonic acid 45.859 33.11 61.2 Glucuronic acid 49.475 0.259 0.2

[0060] Using dextran as standard control, the polysaccharide content of Chitosan fragrans was determined by sulfuric acid phenol method, and the total polysaccharide content was 93.96%.

[0061] Using galacturonic acid as the standard control, the uronic acid content in the chitosan polysaccharide was determined by the carbazole sulfate method, and the uronic acid content was 63.49%.

[0062] Example 3

[0063] This example studies Example 1 to prepare Chitosan polysaccharide (CSHYP) to enhance the phagocytic activity of macrophages, and the specific steps are as follows:

[0064] MTT method to screen the optimal concentration: RAW264.7 cells, digested and counted, and the cell concentration was adjusted to 2.0×10 5 / mL was inoculated into a 96-well cell culture plate at 100μL per well and the cell suspension was added. After 24h of adherent culture, 100μL of CSHYP dilutions of different concentrations (50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, 800μg / mL) were added, and the cells were incubated in a 37℃ CO2 incubator for 20h. 50μL of 2mg / mL MTT solution was added and incubated for another 4h. The 96-well plate was removed, centrifuged at 2000rpm for 5min, the supernatant was discarded, 150μL of acidic DMSO was added to each well, and the micro-oscillator was shaken for 15min, and the OD value was measured at a wavelength of 490nm. Among them, the control group (0μg / mL) was added with an equal amount of DMEM culture solution.

[0065] Phagocytic activity of macrophages: Take a 24-well plate and inoculate 4×10 RAW264.7 cells 5 / well, and the drug was administered after 2 hours of adhesion. A control group (Ctrl group), a positive control LPS group (100ng / mL), and CSHYP concentration groups (50μg / mL, 100μg / mL, 200μg / mL) were set up. 24 hours after drug administration, the cells were collected and placed in 2 flow tubes, centrifuged at 1500rpm, the supernatant was discarded, and precooled on an ice bath for 1min. After precooling, 100μL of FITC-labeled dextran (1mg / mL) was added to the flow tubes, one group was placed in an ice bath at 4℃ for 30min, and the other group was placed in a 37℃ incubator for 30min. The flow tubes were taken out, 2mL of precooled PBS was added to each tube to terminate the reaction, centrifuged at 1500rpm, the supernatant was discarded, and 2mL of ice-bathed PBS buffer was added to each tube for washing once, and centrifuged at 1500rpm. The supernatant was discarded, and 500μL of PBS buffer was added to resuspend the cells, and the phagocytic activity was detected by flow cytometry. The difference of the mean fluorescence intensity of three times of each sample at 4°C and 37°C was calculated to obtain ⊿MFI=MFI(37°C)-MFI(4°C). The control group was DMEM culture medium, and each concentration group of CSHYP was prepared by dissolving the chitosan polysaccharide prepared in Example 1 in DMEM culture medium and then diluting it.

[0066] The results are as follows Figure 4 The results showed that the CSHYP group had no cytotoxicity within the concentration of 200 μg / mL ( Figure 4 A); the positive control LPS group and the CSHYP concentration groups (50μg / mL, 100μg / mL, 200μg / mL) significantly increased the phagocytic activity of macrophages (P<0.001) ( Figure 4 B), indicating that all concentration groups of Chitosan CSHYP have immune enhancement activity.

[0067] Example 4

[0068] This example studies the effect of the chitosan polysaccharide CSHYP prepared in Example 1 on promoting the release of inflammatory factors in macrophages, and the specific steps are as follows:

[0069] Take a 24-well plate and inoculate 2×10 RAW264.7 cells 5 / well, adhere to the wall and culture for 24 hours before administration, set up a control group (Ctrl group), a positive control LPS group (100ng / mL), and CSHYP concentration groups (50μg / mL, 100μg / mL, 200μg / mL), placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, and collect the cell supernatant. ELISA kits and NO kits were used to detect the content of IL-1β, IL-10, IL-6, TNF-α and NO in the cell supernatant. Among them, the control group was DMEM culture medium, and the CSHYP concentration groups were prepared by dissolving the Quzhi chitosan polysaccharide prepared in Example 1 in DMEM culture medium and then diluting it.

[0070] The results are as follows Figure 5 The results showed that the positive control LPS group and each concentration group of CSHYP (50μg / mL, 100μg / mL, 200μg / mL) significantly promoted the release of macrophage inflammatory factors IL-1β, IL-10, IL-6, TNF-α and NO (P<0.001), indicating that each concentration group of Quzhi Chitosan CSHYP improved immune activity by promoting the release of macrophage inflammatory factors.

[0071] Example 5

[0072] This example studies the effect of the chitosan polysaccharide CSHYP prepared in Example 1 on promoting the release of ROS from macrophages, and the specific steps are as follows:

[0073] Take a 24-well plate and inoculate 8×10 RAW264.7 cells per well. 6, 2h after attachment, drug administration, control group (Ctrl group), positive control LPS group (100ng / mL), CSHYP concentration groups (50μg / mL, 100μg / mL, 200μg / mL) were set up respectively. 4h after administration, cells were collected, centrifuged at 1000rpm for 5min (4℃), supernatant was discarded, 1mL DMEM culture medium was added for washing, centrifuged at 1000rpm for 5min (4℃), supernatant was discarded. Then 1mL DCFH-DA (10μM) was added for re-suspending, and placed in a 37℃ constant temperature incubator for incubation for 30min. Centrifuged at 1000rpm for 5min (4℃), supernatant was discarded, 1mL pre-cooled DMEM culture medium was added to each tube for washing twice, centrifuged at 1000rpm for 5min, supernatant was discarded, 500μL PBS buffer was added for re-suspending cells and transferred to flow tubes, and the intensity of fluorescence was detected by flow cytometry at 488nm excitation wavelength and 525nm emission wavelength to calculate the amount of ROS released. The control group was DMEM culture medium, and each concentration group of CSHYP was prepared by dissolving the Chitosan polysaccharide prepared in Example 1 in DMEM culture medium and then diluting it.

[0074] The results are as follows Figure 6 The results showed that the positive control LPS group and each concentration group of CSHYP (50μg / mL, 100μg / mL, 200μg / mL) significantly promoted the release of ROS in macrophages (P<0.001), indicating that each concentration group of Quzhi Chitosan CSHYP improved immune activity by promoting the release of ROS in macrophages.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chitosan polysaccharide, characterized in that: In terms of molar percentage, the monosaccharide composition of the chitosan polysaccharide is 60.2-62.2% of galacturonic acid, 13.6-15.6% of arabinose, 19.0-21.0% of galactose, 2.5-3.5% of rhamnose, 0.3-0.5% of mannose, 0.2-0.4% of fucose, 0.2-0.4% of glucuronic acid, 0.05-0.15% of glucose and 0.05-0.15% of xylose.

2. The chitosan polysaccharide according to claim 1, characterized in that Taking dextran as the standard reference, the total polysaccharide content of the Quzhizhi polysaccharide is 90.96-96.96%; taking galacturonic acid as the standard reference, the uronic acid content of the Quzhizhi polysaccharide is 60.49-66.49%.

3. The method for preparing the chitosan polysaccharide according to claim 1 or 2, characterized in that: The steps include: Extracting the Fructus Aurantii Immaturus with water to obtain a Fructus Aurantii Immaturus water extract; concentrating the Fructus Aurantii Immaturus water extract under reduced pressure and subjecting it to alcohol precipitation to obtain a precipitate; re-dissolving the precipitate with water, performing chromatography, elution, concentrating under reduced pressure, and freeze-drying to obtain a crude polysaccharide of Fructus Aurantii Immaturus; The crude polysaccharide of Quzhizi is enzymatically hydrolyzed to obtain an enzymatic hydrolyzate; Sevage solution is added to the enzymatic hydrolyzate for deproteinization; after the deproteinization, the water layer is placed in a dialysis bag for dialysis, and freeze-dried to obtain the polysaccharide of Quzhizi.

4. The preparation method according to claim 3, characterized in that: During water extraction, the solid-liquid ratio of Quzhi Fructus and water is 1g:8mL~1g:12mL.

5. The preparation method according to claim 3, characterized in that: The water extract of Fructus Aurantii Immaturus is concentrated under reduced pressure to 5% to 10% of the original volume.

6. The preparation method according to claim 3, characterized in that: The adsorption material of the chromatography is AB-8 macroporous adsorption resin.

7. The preparation method according to claim 3, characterized in that: The enzyme for enzymolysis is papain; the conditions for enzymolysis are enzymolysis in a water bath at 55-65° C. for 0.5-2 h.

8. The preparation method according to claim 3, characterized in that: The specification of the dialysis bag is a dialysis bag with a molecular weight cut-off of 3500Da.

9. Use of the chitosan polysaccharide according to claim 1 or 2 in the preparation of medicines or health foods for enhancing immunity.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Chitosan polysaccharide according to claim 1 or 2 and pharmaceutically acceptable excipients.

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