Uniform acanthopanax water-soluble glucan as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant active polysaccharides, and particularly relates to uniform acanthopanax water-soluble glucan as well as a preparation method and application thereof. The weight-average molecular weight of the acanthopanax water-soluble glucan is 33.31 kDa, and the acanthopanax water-soluble glucan has a branched chain structure. The acanthopanax senticosus water-soluble glucan can be obtained by deproteinizing an ethanol precipitate obtained in an acanthopanax senticosus injection process and separating the ethanol precipitate with a DEAE-52 cellulose column and a SephadexG-100 gel column, and is newly found glucan. Experiments show that the acanthopanax water-soluble glucan has a remarkable tumor inhibition effect on CT26.WT tumor-bearing mice, can protect immune organs, and can be used for preparing immunopotentiators, especially anti-tumor immunopotentiators including drugs, health-care foods and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of plant active polysaccharides, and particularly relates to a uniform Acanthopanax senticosus water-soluble glucan and a preparation method and application thereof. Background Art

[0002] Acanthopanax senticosus, a traditional herbaceous plant of the genus Acanthopanax in the family Araliaceae, is widely distributed in Northeast and North my country, Japan, South Korea, and the Russian Far East. Acanthopanax has a long history and wide application in the field of traditional Chinese medicine due to its rich bioactive ingredients, such as glycosides, polysaccharides, flavonoids, phytic acids, and a variety of nutrients. In recent years, with the deepening of research on natural medicines, Acanthopanax senticosus polysaccharides have attracted widespread attention in the scientific research community due to their unique pharmacological effects and potential anti-tumor activity. However, this "precious" resource has not yet been reasonably utilized. The alcohol precipitate containing a large amount of polysaccharides produced during the production of Acanthopanax injection is discarded as waste, which not only causes environmental pollution, but also causes serious waste of resources.

[0003] At present, there are fewer research reports on Acanthopanax polysaccharides compared to other polysaccharides. Due to the complexity of the physical and chemical properties of polysaccharides, although water extraction and alcohol precipitation, Sevage method for protein removal, DEAE-52 and Sephadex G-100 purification are commonly used in the preparation of Acanthopanax polysaccharides, the structures and activities of Acanthopanax polysaccharides prepared by different processes vary greatly. Acanthopanax polysaccharides reported in the literature are basically heteropolysaccharides, such as Acanthopanax polysaccharides reported by Tong Xiaoyu, Bai Yeming, etc. (Northeast Normal University degree thesis: "Isolation, Purification and Structural Research of Acanthopanax Polysaccharides", Heilongjiang University degree thesis: "Extraction, Purification and In Vitro Anti-Lung Cancer Activity of Acanthopanax Fruit Polysaccharides"), Acanthopanax polysaccharides disclosed in CN202010733740.1, etc.

[0004] At present, there is a lack of detailed reports on the fine chemical structure of Acanthopanax senticosus polysaccharides with anticancer activity, and a suitable extraction and purification process has not yet been developed to prepare Acanthopanax senticosus polysaccharides with anticancer activity with clear chemical structure and uniform composition. These factors limit the research and development of new anticancer drugs of Acanthopanax senticosus polysaccharides. Summary of the invention

[0005] In view of the above problems, the present invention unexpectedly discovered a uniform water-soluble glucan with high oral immune enhancement and tumor inhibition effects during the process of extracting polysaccharides from ethanol sediments in the preparation process of Acanthopanax senticosus injection. Therefore, the purpose of the present invention is to provide an Acanthopanax senticosus water-soluble glucan and its preparation method and use.

[0006] The technical solutions of the present invention are as follows:

[0007] First, the present invention provides an Acanthopanax senticosus water-soluble glucan, which is α-glucan. Chromatographic analysis shows that the polysaccharide is a homogeneous polysaccharide. The Acanthopanax senticosus water-soluble glucan is composed of Glcp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→, →3,4)-Glcp-(1→, →4,6)-Glcp-(1→ residues in a molar ratio of 2.90:10.76:1.00:1.06:1.83; the main chain of the Acanthopanax senticosus water-soluble glucan is composed of Glcp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→, →3,4)-Glcp-(1→, →4,6)-Glcp-(1→ residues; the weight average molecular weight Mw of the Acanthopanax senticosus water-soluble glucan is 33.31 kDa.

[0008] Secondly, the present invention provides a method for preparing the aforementioned Acanthopanax senticosus water-soluble glucan, comprising the following steps:

[0009] In the following steps, ethanol concentration and ethanol content are both volume fractions.

[0010] S1: dissolving the ethanol precipitate obtained in the process of Acanthopanax senticosus injection in water, adding ethanol to the precipitate aqueous solution for re-precipitation, re-dissolving the precipitate for re-precipitation, removing protein by Sevage method, removing small molecule impurities by dialysis, and freeze-drying to obtain Acanthopanax senticosus crude polysaccharide;

[0011] S2: Take crude polysaccharide of Acanthopanax senticosus, add water to make a solution with a mass percentage of 2-6%, load it onto a DEAE-52 cellulose column, elute with ultrapure water, and the elution rate is 1-5 mL / min. The eluted polysaccharide solution is concentrated, dialyzed at 4°C, with a molecular weight cutoff of 3.5 kDa (i.e., dialyzed at 4°C using a dialysis bag with a molecular weight cutoff of 3500 Da), and the retained solution is freeze-dried, and the eluted product is taken as the separated sugar of Acanthopanax senticosus;

[0012] S3: Take the Acanthopanax senticosus separated sugar, add water to dissolve it, load it onto a Sephadex G-100 gel column, elute with ultrapure water, and the elution rate is 0.5-1.5 mL / min. Collect the main components according to the elution curve, dialyze at 4°C, with a molecular weight cutoff of 3.5 kDa (i.e., dialyze at 4°C using a dialysis bag with a molecular weight cutoff of 3500 Da), and lyophilize to obtain the Acanthopanax senticosus water-soluble glucan.

[0013] Preferably, after the first alcohol precipitation in step S1, the precipitate is dissolved in water at a solid-liquid ratio of 1:160, the precipitate aqueous solution is concentrated under reduced pressure to 1 / 5 of the original volume, ethanol is slowly added with continuous stirring to make the ethanol concentration reach 75-85%, and the mixture is allowed to stand at 4°C overnight and freeze-dried to obtain the Acanthopanax senticosus crude polysaccharide alcohol precipitate.

[0014] Preferably, the method for removing protein by Sevage method in step S1 comprises the following steps: preparing the Acanthopanax senticosus crude polysaccharide alcohol precipitation with water into an Acanthopanax senticosus crude polysaccharide alcohol precipitation aqueous solution with a concentration of 20 mg / mL; preparing chloroform and n-butanol into a mixed organic reagent in a volume ratio of 4:1, mixing the mixed organic reagent with the Acanthopanax senticosus crude polysaccharide alcohol precipitation aqueous solution in a volume ratio of 1:1, shaking for 30 minutes, centrifuging the mixed solution at 4000 rpm for 15 minutes, and collecting the uppermost sugar solution. Further preferably, in the step of removing protein by Sevage method, the sugar solution continues to be deproteinized with chloroform and n-butanol mixed organic reagent, and the deproteinization is performed 9 times in total.

[0015] Preferably, in steps S2 and S3, the dialysis time is 48 hours, and the distilled water is changed every 2 to 3 hours.

[0016] The ethanol precipitate obtained in the process of Acanthopanax Senticosus Injection can be obtained from the Acanthopanax Senticosus Injection manufacturer or prepared by oneself, and can be prepared by oneself using the following method:

[0017] Take an appropriate amount of Acanthopanax senticosus powder, add 4 to 8 times the weight of distilled water of Acanthopanax senticosus powder, and soak for 20 to 40 minutes. Extract for 1 hour under a slightly boiling state, release the extract, add 4 to 8 times the amount of distilled water, and continue to extract for 4 hours. Combine the two extracts. When the temperature of the extract drops below 40°C, adjust the pH to 10 to 12 with lime milk, and maintain the pH for 0.5 hours. Then adjust the pH value of the extract to 5 to 6 with 20% sulfuric acid solution and let it stand for more than 5 hours. Concentrate the supernatant under reduced pressure to a relative density of 1.10 to 1.13 (80°C). When the temperature of the concentrated solution drops below 40°C, slowly add ethanol under stirring to make its ethanol concentration reach 83%, and let it stand for more than 12 hours after sufficient stirring. Perform high-speed centrifugation, and the supernatant is used for the preparation of Acanthopanax injection. The ethanol precipitate obtained by centrifugation is dried under reduced pressure for subsequent preparation of polysaccharides.

[0018] Again, the present invention provides the use of the Acanthopanax senticosus water-soluble glucan and the Acanthopanax senticosus water-soluble glucan prepared by the above preparation method in the preparation of an immunopotentiator. The immunopotentiator can increase the organ coefficient of animal immune organs and increase the levels of TNF-α, IL-2, and IFN-γ in animal blood.

[0019] Preferably, the immunopotentiator is an anti-tumor immunopotentiator, that is, the immunopotentiator can inhibit tumor growth. The tumor is preferably colorectal cancer.

[0020] Preferably, the immunopotentiator is an oral preparation, that is, the immunopotentiator still has the activity of enhancing immunity and inhibiting tumor growth after oral gastrointestinal metabolism and enzymatic hydrolysis.

[0021] The beneficial effects of adopting the above scheme include:

[0022] (1) The Acanthopanax senticosus water-soluble glucan of the present invention is prepared from ethanol precipitate waste in the preparation of Acanthopanax senticosus injection, thereby improving the utilization rate of traditional Chinese medicine resources and reducing environmental pollution.

[0023] (2) The Acanthopanax senticosus water-soluble glucan of the present invention has uniform composition, high immune enhancement and tumor inhibition activity, and protective effect on immune organs, and can be used to prepare anti-tumor drugs, immunomodulatory drugs and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The molecular weight chromatogram of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0025] Figure 2 This is a chromatogram of the monosaccharide composition of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0026] Figure 3 The Fourier transform infrared spectrum (FT-IR) of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0027] Figure 4 This is a scanning electron microscope (SEM) image of the Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0028] Figure 5 This is a methylation GC-MS chromatogram of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0029] Figure 6 This is the nuclear magnetic resonance 1H spectrum of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0030] Figure 7 This is the nuclear magnetic resonance 13C spectrum of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0031] Figure 8 The nuclear magnetic resonance COSY spectrum of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0032] Fig. 9 The nuclear magnetic resonance HSQC spectrum of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0033] Fig.10 This is the nuclear magnetic resonance HMBC spectrum of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention;

[0034] Fig.11 The effect of Acanthopanax senticosus polysaccharide ASPN-1 on the tumor weight of each group;

[0035] Fig.12 This is a graph showing the tumor inhibition rate of Acanthopanax senticosus polysaccharide ASPN-1 on CT26.WT sarcoma;

[0036] Fig.13 This is a graph showing the effect of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention on the thymus and spleen indexes of CT26.WT tumor-bearing mice;

[0037] Fig.14 This is a graph showing the effect of Acanthopanax senticosus polysaccharide ASPN-1 of the present invention on the levels of IL-2, TNF-α and IFN-γ in the serum of tumor-bearing mice;

[0038] In the figure: * Indicates P < 0.05 compared with the blank group; # Indicates P < 0.05 compared with the model group. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearly and completely presented, the technical scheme of the present invention will be described in detail in combination with the embodiments of the present invention. The provided embodiments are only some implementation cases of the present invention and are not regarded as limiting the scope of protection of the present invention. In the following embodiments, Acanthopanax senticosus polysaccharide ASPN-1 is Acanthopanax senticosus water-soluble glucan.

[0040] In the following examples, ethanol concentration and ethanol content are both volume fractions.

[0041] The preparation method of the ethanol precipitate obtained in the Acanthopanax injection process used in the following example is as follows. Take an appropriate amount of Acanthopanax medicinal material powder, add 4 to 8 times the weight of distilled water, and soak for 20 to 40 minutes. Extract for 1 hour under a slightly boiling state, release the extract, and then add 4 to 8 times the amount of distilled water, and continue to extract for 4 hours. Combine the two extracts. When the temperature of the extract drops below 40°C, adjust the pH to 10 to 12 with lime milk, and maintain the pH for 0.5 hours. Then adjust the pH value of the extract to 5 to 6 with 20% sulfuric acid solution and let it stand for more than 5 hours. The supernatant is concentrated under reduced pressure to a relative density of 1.10 to 1.13 (80°C). When the temperature of the concentrated solution drops below 40°C, slowly add ethanol under stirring to make its ethanol concentration reach 83%, and let it stand for more than 12 hours after sufficient stirring. Perform high-speed centrifugation, and the ethanol precipitate obtained by centrifugation is dried under reduced pressure for subsequent preparation of polysaccharides.

[0042] Example 1: Extraction, separation and purification of Acanthopanax senticosus polysaccharides

[0043] (1) Preparation of Acanthopanax senticosus polysaccharide

[0044] The ethanol precipitate obtained in the Acanthopanax injection process was crushed, 25 g of the alcohol precipitate powder was taken into a beaker, 4000 mL of distilled water was added to the beaker at a solid-liquid ratio of 1:160, and the mixture was stirred at 90°C for 2 h to fully dissolve. The mixture was centrifuged at 8000 rpm for 15 min, and the supernatant was concentrated under reduced pressure to 1 / 5 of the original volume. Ethanol was slowly added to make the ethanol concentration in the solution reach 80%, and the solution was allowed to stand at 4°C overnight and freeze-dried to obtain the Acanthopanax polysaccharide extract.

[0045] Acanthopanax senticosus polysaccharide extract was prepared into a 20 mg / mL solution with water, and the protein impurities were removed by Sevage method. Chloroform and n-butanol were first prepared into Sevage reagent in a volume ratio of 4:1. Then, the Sevage reagent was mixed with the Acanthopanax senticosus crude polysaccharide solution in a volume ratio of 1:1 and vigorously shaken for 30 minutes. The mixed solution was centrifuged (4000rpm, 15min), the sugar solution on the top layer was collected, and the white protein in the middle layer was discarded. Sevage reagent was added 9 times to remove all proteins in the sample. After the protein was removed, the Acanthopanax senticosus crude polysaccharide solution was concentrated to 1 / 5 of the original volume under reduced pressure and then transferred into a dialysis bag with a molecular weight cutoff of 3.5kDa. It was dialyzed at 4°C for 48 hours, and the distilled water was changed every 2 to 3 hours. The dialysis intercept was freeze-dried to obtain the dry Acanthopanax senticosus crude polysaccharide (ASP).

[0046] (2) Separation of Acanthopanax senticosus polysaccharides using DEAE-52 cellulose chromatography column

[0047] Accurately weigh 1g of the above-mentioned Acanthopanax senticosus crude polysaccharide dry product (ASP) and fully dissolve it in 50mL ultrapure water. Use a rubber-tipped dropper to drop all the prepared crude polysaccharide solution into a DEAE-52 cellulose anion exchange chromatography column (5.0×60cm), and elute with ultrapure water, 0.3 and 0.5mol / LNaCl solution in turn, and collect with an automatic collector. The elution flow rate is 3mL / min, 13min / tube. Pipette 200μL of sugar solution in the collection tube, mix it evenly with 200μL of ultrapure water and 200μL of 5% phenol solution, and then add 1mL of concentrated sulfuric acid. After shaking well, let it stand for 10min, take 200μL of reaction solution from each tube into a 96-well plate, place it in a FlexA-200 microplate reader to measure the absorbance at 490nm, and make an elution curve based on the absorbance. Collect polysaccharide components in different peak segments according to the elution curve. The different eluted fractions were transferred into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in distilled water at 4°C for 48 hours, with the distilled water changed every 2 to 3 hours. After the dialysis was completed, vacuum freeze-dried to obtain three fractions: ASPN (water elution), ASPA-1 (0.3 M NaCl solution elution), and ASPA-2 (0.5 M NaCl solution elution).

[0048] (3) Gel column chromatography purification of Acanthopanax senticosus neutral polysaccharide ASPN

[0049] Weigh 20 mg of the dry product of Acanthopanax neutral sugar ASPN and dissolve it in 4 mL of ultrapure water, centrifuge at 8000 rpm for 10 min, and load the supernatant onto a Sephadex G-100 chromatography column (2.6 cm × 120 cm) filled with dextran gel. The elution conditions are: eluent: ultrapure water; flow rate: 0.6 mL / min, 10 min / tube. Collect with an automatic collector. Then use the phenol-sulfuric acid method to determine the sugar content and draw the elution curve. According to the elution curve results, collect the main components, and the purified polysaccharide solution is concentrated under reduced pressure, transferred into a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in 4°C distilled water for 48 hours, changing the distilled water every 2 to 3 hours. After the dialysis is completed, vacuum freeze-dry. Acanthopanax water-soluble glucan ASPN-1 is obtained.

[0050] Example 2: Physicochemical properties and structural characterization of Acanthopanax senticosus water-soluble glucan ASPN-1

[0051] (1) Determination of molecular weight of Acanthopanax senticosus glucan ASPN-1

[0052] HPGPC was used to determine the homogeneity and molecular weight of Acanthopanax senticosus water-soluble glucan ASPN-1. Acanthopanax senticosus water-soluble glucan ASPN-1 was prepared into a sample solution with a concentration of 2.0 mg / mL using ultrapure water, and the prepared solution was filtered through a 0.22 μm filter membrane before testing.

[0053] Chromatographic conditions: Chromatography: High performance gel permeation chromatograph (Agilent 1260, USA); Detector: Differential refractive index detector (RID, G1362A, 40°C); Chromatographic column: Waters Ultrahydrogel TM Gel column (TM120, TM250, TM500; 7.8×300mm); mobile phase: 0.1mol / LNaNO3; flow rate: 1mL / min; column temperature: 40℃; running time: 35min. The molecular weight of Acanthopanax senticosus water-soluble glucan ASPN-1 was calculated using the T series dextran standard curve equation.

[0054] like Figure 1 As shown, the HPGPC spectrum of Acanthopanax senticosus water-soluble glucan ASPN-1 shows that it is a single symmetrical peak, indicating that Acanthopanax senticosus water-soluble glucan ASPN-1 has good molecular uniformity. The weight average molecular weight (Mw) of ASPN-1 is 33.31 kDa.

[0055] (2) Analysis of monosaccharide composition of Acanthopanax senticosus water-soluble glucan ASPN-1

[0056] The monosaccharide composition of Acanthopanax senticosus water-soluble glucan ASPN-1 was determined by PMP pre-column derivatization HPLC.

[0057] Acid hydrolysis: Accurately weigh the polysaccharide in a small glass bottle, add 1 mL of ultrapure water and 1 mL of 4 M trifluoroacetic acid solution in turn, dissolve and mix evenly, then fill with nitrogen and seal the tube, and hydrolyze at 110°C for 2 h; after complete hydrolysis, take 0.1 mL of the cooled hydrolyzate and dry it at 60°C for 2 h.

[0058] Pre-column derivatization: The above sample was dissolved in 0.05 mL of 0.3 M sodium hydroxide solution and 0.05 mL of PMP (i.e., 1-phenyl-3-methyl-5-pyrazolone)-methanol solution, and then nitrogen was filled again and transferred to a 70°C metal bath for reaction for 60 min; then, the reaction system was immediately cooled and mixed evenly with 0.5 mL of 0.3 M hydrochloric acid solution, at which point the derivatization reaction was completed. Next, 0.75 mL of ultrapure water and 1.5 mL of chloroform were added, and after sufficient shaking and mixing, the mixture was transferred to a separatory funnel for extraction, and the water layer was taken and the organic reagent layer was discarded; the extraction process was repeated three times, and the final water layer liquid was collected for on-machine detection.

[0059] HPLC detection: The aqueous layer solution obtained in the derivatization and the prepared monosaccharide mixed standard solution were filtered through a 0.22μm filter membrane and then subjected to HPLC analysis. The chromatographic conditions are as follows: the chromatographic column is a Dikma C18 chromatographic column (4.6mm×250mm×5μm), the injection volume is 10μL, the column temperature is 25℃, the mobile phase speed is 1mL / min, the mobile phase A is 0.1M, Na2HPO4 and NaH2PO4 buffer solution, the mobile phase B is acetonitrile, and the isocratic elution is performed in the ratio of A:B=83:17. The detector used is an ultraviolet detector. Finally, the absorbance values ​​of the standard and sample are detected at 254nm, and the signal conversion is performed. According to the difference in peak time and peak area, the monosaccharide composition and proportion in the polysaccharide sample can be determined. The results are as follows Figure 2 As shown, ASPN-1 is composed of glucose and thus is judged to be a glucan.

[0060] (3) Identification of characteristic functional groups of Acanthopanax senticosus water-soluble glucan ASPN-1 by infrared spectroscopy

[0061] 2 mg of ASPN-1 polysaccharide was accurately weighed and mixed with 150 mg of potassium bromide (KBr), crushed and pressed into tablets with a thickness of 1.5 mm. The pressed samples were then scanned using a Fourier transform infrared spectrometer (Nicolet iS5, Thermo) to analyze the characteristic absorption peaks in the polysaccharide sample. The scanning range was 4,000-400 cm -1 , the number of scans is 32 times.

[0062] The results are as follows Figure 3 As shown, at 3385cm -1The broad and strong absorption peak at about 2929cm -1 and 1421cm -1 The absorption band at 1640cm is attributed to CH stretching vibration and CH angle vibration. -1 The absorption near 1154cm corresponds to the stretching vibration of carboxyl-bound water. -1 、1078cm -1 and 1020cm -1 The three characteristic absorption peaks at 943 cm-1 are related to the stretching vibration of the COC glycosidic bond in the pyranose ring. Similarly, the analysis further shows that -1 and 762cm -1 A unique absorption band was observed at 846 cm-1, indicating the presence of a pyranose ring. Importantly, -1 The peak of ASPN-1 was observed at , indicating the presence of an α-configuration glycosidic bond.

[0063] (4) Scanning electron microscopy (SEM) analysis of ASPN-1 showed that the morphological characteristics

[0064] 5 mg of ASPN-1 sample was attached to the sample platform, and a layer of gold powder was coated on the sample by ion sputtering to make it a conductor. Finally, the morphological characteristics of ASPN-1 were observed using a Zeiss Sigma 500 scanning electron microscope (SEM). The vacuum acceleration voltage was 5.0 kV.

[0065] The results are as follows Figure 4 As shown in the figure, the surface morphology of ASPN-1 observed by scanning electron microscopy at 500 times (scale: 20 μm) and 3000 times (scale: 4 μm) magnification is typical of amorphous powder, showing an irregular network structure composed of a large number of flaky spherical particles (0.5-4 μm) and curled filamentous structures. In addition, many tiny holes or grooves were observed on the spherical particles of ASPN-1.

[0066] (5) Methylation analysis of ASPN-1 glycosidic bond composition

[0067] After methylation, hydrolysis and acetylation, the ASPN-1 sample was determined by GC-MS and compared with the standard mass spectrum library. Weigh the polysaccharide sample (3 mg) and place it in a glass reaction bottle, add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction. Take the methylated polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) to hydrolyze for 90 minutes, and evaporate to dryness on a rotary evaporator. Add 2 ml of double distilled water to the residue, reduce 60 mg of sodium borohydride for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry in an oven at 101°C, then add 1 ml of acetic anhydride to acetylate at 100°C for 1 hour, and cool. Then add 3 mL of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4-5 times to remove excess acetic anhydride. The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, and the upper aqueous solution was removed. This was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, fixed to 10 mL, and placed in a liquid phase vial. The acetylated product sample was analyzed using an Agilent GCMS6890-5973 gas chromatograph-mass spectrometer;

[0068] GC-MS conditions: Shimadzu RXI-5SILMS column 30m*0.25mm*0.25um; programmed temperature conditions: starting temperature 120°C, heating at 3°C / min to 250°C / min; maintaining for 5 min; injection port temperature 250°C, detector temperature 250°C / min, carrier gas helium, flow rate 1 mL / min.

[0069] The results are as follows Figure 5 As shown, ASPN-1 is composed of Glcp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→, →3,4)-Glcp-(1→, →4,6)-Glcp-(1→ residues with a molar ratio of 2.90:10.76:1.00:1.06:1.83. The results showed that ASPN-1 is mainly composed of →4)-Glcp-(1→ residues, accounting for about 61.31%, and is bifurcated at O-3 and O-6. The terminal residue content of Glcp-(1→, (~16.52%) reflects the content of two breakpoints in ASPN-1→3,4)-Glcp-(1→, combined with→4,6)-Glcp-(1→, (~16.46%), indicating that the number of terminal sugars in ASPN-1 is equivalent to the number of branch points. It is worth noting that we found 3,4)-Glcp-(1→ residues in Acanthopanax senticosus polysaccharide, a structure that rarely appears in natural glucans.

[0070] Table 1 Analysis of results of Acanthopanax senticosus water-soluble glucan ASPN-1 methylated sugar alcohol acetyl ester (PMAA)

[0071]

[0072] (6) NMR analysis of ASPN-1 glycosidic bond connection mode and connection order

[0073] In the NMR analysis, the deuterated ASPN-1 sample (60 mg) was dissolved in 0.6 mL of D2O (99.9 atomic % D, Adamas β), and the supernatant was collected by centrifugation (10000 rpm, 8 min) and then subjected to NMR analysis. All NMR experiments, including those of ASPN-1 1 H. 13 C NMR, 1H-1H correlation spectra ( 1 H- 1 H COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra were all performed on a Bruker Advance III 600 MHz NMR spectrometer at 298 K.

[0074] ASPN-1 1 H spectrum Figure 6 As shown, in the anomeric hydrogen region (δ4.5~5.5ppm), there are mainly 7 anomeric proton signals, and their chemical shifts are 5.38ppm, 5.34ppm, 5.31ppm, 4.96ppm, 4.93ppm, 5.21ppm and 4.63ppm respectively. The chemical shifts of the anomeric carbon corresponding to these anomeric hydrogens can be 13 C spectrum ( Figure 7 ) and HSQC spectra ( Fig. 9 ) were assigned and seven residues were identified: A(δ H / C 5.38 / 99.44ppm), B(δ H / C 5.34 / 99.66ppm), C(δ H / C 5.31 / 98.53ppm), D(δ H / C 4.96 / 97.60ppm), E(δ H / C 4.93 / 98.50ppm), R α (δ H / C 5.21 / 91.80ppm) and R β (δ H / C 4.63 / 95.68ppm). After the head area signal is determined, 1 H NMR, 13 C NMR, 1 H / 1H COSY, HSQC correlation spectra, all the main sugar residues 1 H and 13 The C signals were assigned respectively, and the results are shown in Table 2.

[0075] Table 2 Assignment of NMR chemical shifts of ASPN-1 in D2O (δ 4.79) at 298K.

[0076]

[0077] Residue A 1 The H spectrum shows that the peak intensity of the anomeric proton region is the highest at δ5.38ppm. 13 The chemical shift of C spectrum is δ C 99.94 ppm, indicating that residue A is a typical α configuration. Based on the anomeric region A H1-C1 (δ H / C 5.38 / 99.44), passed HSQC, 1 H / 1 H COSY analysis gave A H2-C2(δ H / C 3.60 / 71.31)、A H3-C3(δ H / C 3.95 / 73.31)、A H4-C4(δ H / C 3.65 / 76.73)、A H5-C5(δ H / C 3.89 / 70.12) and A H6 a,b -C6(δ H / C 3.83,3.75 / 60.38). The chemical shift of A C4 (δ C 76.73) shifted to the downfield, indicating that a substitution occurred at the C-4 position. Combining the literature and methylation analysis, it was preliminarily inferred that residue A was clearly the main chain of ASPN-I →4)-α-D-Glcp-(1→.

[0078] The anomeric region of residue B, B H1-C1 (δ H / C 5.34 / 99.66). 1 H / 1 In the H COSY spectrum, the chemical shifts of the protons in residue B were determined: H2(δ H 3.56ppm), H3(δ H 3.93ppm), H4(δ H 3.61ppm), H5(δ H 3.82ppm) and H6(δ H 3.40ppm), and the corresponding carbon signals were successfully obtained by HSQC, which were the δ C71.57, 73.16, 76.41, 71.04 and 69.18. Among them, C4 (δ C 76.41) and C-6 (δ C 69.18) two carbon signals shifted to the low field, combined with the methylation results, residue B was determined to be →4,6)-α-D-Glcp-(1→. According to HSQC, 1 H / 1 H COSY and HMBC analysis revealed that the 1 H and 13 C chemical shift in C H1-C1( H / C 3.51 / 99.53)、C H2-C2(δ H / C 3.61 / 72.80), CH3-C3(δ H / C 3.96 / 76.75)、C H4-C4(δ H / C 3.60 / 76.63)、C H5(δ H 3.88) and C H6 a,b -C6(δ H / C 3.83,3.75 / 60.38). Among them, C3(δ C 76.75) and C4(δ C 76.63) The two carbon signals shifted to the low field. Combining the literature and methylation results, residue C was determined to be →3,4)-α-D-Glcp-(1→. Similarly, residues D and E were identified as α-D-Glcp-(1→, →6)-α-D-Glcp-(1→.

[0079] In addition to the residues AE, there are also chemical shift values ​​δ H The two peaks at 5.21 and 4.63 ppm represent two specific sugar residues R α and R β ( Figure 6 ), the chemical shift of the anomeric carbon of these two sugar residues in HSQC is δ C 91.80 and 95.68 ppm ( Fig. 9 ). α and R β Represents α and β reducing terminal glucose residues.

[0080] To further investigate the glycosyl linkage sequence, the long-range correlation HMBC spectrum of ASPN-1 was analyzed ( Fig.10). HMBC cross peaks were used to elucidate the connectivity between glucose moieties, as summarized in Table 3. The HMBC spectra showed strong cross peaks at 5.38 / 76.73ppm (A H1-A C4) and 3.65 / 99.44ppm (A H4-A C1), indicating the presence of the dominant sequence [→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→]. The cross peaks at 5.34 / 76.73ppm (B H1-AC4) and 3.61 / 99.44ppm (BH4-A C1) indicated that the anomeric hydrogen of residue A was connected to O-4 of residue B, indicating the connectivity was [→4)-α-D-Glcp-(1→4,6)-D-Glcp-(1→]. Similarly, the H1-C C4 (5.38 / 76.63ppm) and C H1-A The cross-correlation signals of the heterotopic proton and carbon at C1 (3.60 / 99.44 ppm) indicated that residue A was linked to O-4 of residue C, confirming the existence of the fragment [→3,4)-α-DGlcp-(1→4)-α-D-Glcp-(1→]. The results showed that ASPN-1 has a main chain structure of [→4)-α-D-Glcp-(1→] with branches at residues C-3 and C-6.

[0081] Table 3 Attribution of cross peaks in the HMBC spectrum of ASPN-1.

[0082]

[0083] The cross peaks observed at δ 4.96 / 69.18 (D H1-B C6) and 4.93 / 69.18 (E H1-B C6) indicated that the C-6 position of the residue →4,6)-α-D-Glcp-(1→) was linked to the α-D-Glcp-(1→) residue and / or to the →6)-α-D-Glcp-(1→) residue. The weak cross peaks at 4.93 / 65.42 (E H1-E C6) and 4.96 / 65.42 (D H1-E C6) indicated that the fragment α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→) was The cross peak at C1) indicates that the residue →3,4)-α-D-Glcp-(1→ terminates at C-3 with an α-D-Glcp-(1→ residue. NMR data show that ASPN-1 consists of a main chain composed of →4)-α-D-Glcp-(1→, and the branch end at O-3 is α-D-Glcp-(1→. The branch end at O-6 is composed of α-D-Glcp-(1→ and / or α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→. Methylation analysis showed that the molar ratio of residues A, B, C, D and E is approximately (6:21:2:2:4). Therefore, the putative structure of ASPN-1 is shown in Formula I below:

[0084]

[0085] Example 3: Antitumor activity of Acanthopanax senticosus glucan ASPN-1

[0086] (1) Grouping and drug administration

[0087] Six-week-old male BALB / c mice (weight 20±2g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and housed in a standard experimental environment (relative humidity: 50±5%; ambient temperature: 23±2℃; light: 12L:12D). The test mice underwent a one-week pre-experimental acclimation phase, including daily oral administration of 0.2mL of normal saline to the test mice. After 7 days of adaptation to the experimental conditions, the mice were randomly divided into five groups, each with 8 mice, as follows: blank group, model group, and three ASPN-1 treatment groups (ASPN-1L group: 50mg / kg, ASPN-1M group: 100mg / kg; ASPN-1H group: 200mg / kg) and 5-fluorouracil group (5-Fu, 20mg / kg). CT26.WT mouse colon cancer cells were inoculated subcutaneously in the right forelimb axilla of mice in the model group, ASPN-1 treatment group, and positive control group (approximately 5×10 6The ASPN-1 group was gavaged daily starting from the second day after inoculation, and the positive control group was given 5-Fu at a concentration of 20 mg / kg with an intraperitoneal injection volume of 0.2 mL / mouse, once every two days, until termination. The blank group and the model group were gavaged with normal saline throughout the study, with a volume of 0.2 mL / mouse. Tumor volume changes were measured every 2 days. 18 days after administration, the mice were anesthetized with pentobarbital, and tissues and blood were extracted for subsequent studies.

[0088] (2) Tumor inhibition rate and immune organ index

[0089] The length (L, mm) and width (W, mm) of the tumor were measured with a vernier caliper every other day. 3 )=LW 2 / 2, and draw the tumor volume growth curve of each group. The residual blood on the surface of the cancerous tissue was rinsed three times with cold saline. The exact weight of each tumor was recorded after surgery. Tumor inhibition rate TIR (%) = (WM-WT) / WM × 100%, where WM is the average tumor weight of mice in the model control group, and WT is the average tumor weight of mice in the treatment group ( Fig.11 After the experiment, the weight of the thymus and spleen was accurately recorded. The organ index was then calculated using the following formula: immune organ index (mg / g) = immune organ weight / mouse weight.

[0090] To evaluate the anti-tumor effect of ASPN-1, a mouse colorectal cancer model (CT26.WT) was used for experimental research. Oral ASPN-1 at doses of 50, 100 and 200 mg / kg significantly inhibited tumor growth and reduced tumor weight in a dose-dependent manner (p<0.05), with TIRs of 35.36%, 47.11% and 52.78%, respectively. The TIR of the ASPN-1H group was close to that of the 5-Fu group (66.27%), slightly lower than that of the 5-Fu group. This shows that ASPN-1 has a significant inhibitory effect on the growth of CT26.WT (Table 4, Fig.12 ).

[0091] like Fig.13 As shown in the figure, compared with the blank group, the thymus weight of CT26.WT tumor-bearing mice was significantly reduced, and the spleen weight was significantly increased (p<0.05). These results suggest that the proliferation of CT26.WT tumor cells may impair the function of immune organs. Compared with the model group, the thymus and spleen indexes of mice changed significantly after ASPN-1 administration (p<0.05), indicating that ASPN-1 has the potential to reduce splenomegaly and thymic atrophy, thereby improving the immune dysfunction of CT26.WT tumor-bearing mice. The improvement effect of 5-FU on splenomegaly and thymic atrophy was lower than that of ASPN-1.

[0092] Table 4 Inhibition of tumor growth in CT26.WT colorectal cancer-bearing mice by Acanthopanax senticosus water-soluble glucan ASPN-1

[0093]

[0094] Note: * , compared with the blank group, P<0.05; # , compared with the model group, P<0.05.

[0095] (3) Pathological changes of tumor tissue

[0096] The solid tumors were fixed with neutral polyformaldehyde, embedded in paraffin and further processed. The tissues were then sliced ​​into 4 μm thick slices. HE staining was performed and the pathological changes of the tumor tissues were observed under a Nikon Eclipse Ci-L optical microscope (Nikon, Japan).

[0097] In this study, hematoxylin-eosin (H&E) staining on tumor tissue sections was used to analyze tissue structure and morphological changes. The nucleus was dark blue-purple, and the cytoplasm was pink-purple. The tumor tissue cells in the model group were dense, the nucleus was intact, the nucleolus was clear, the cytoplasm was uniform, and the chromatin was dense, indicating a proliferative environment for tumor cell growth. Compared with the model group, tumor cells in the 5-Fu group and the ASPN-1 treatment group showed necrosis, nuclear fragmentation or dissolution, and the nuclear membrane and nucleolus were blurred. The necrotic area of ​​tumor tissue increased significantly, the cell volume decreased, the cells were loosely arranged, the lymphocyte infiltration increased, and the interstitial space expanded. With the increase in the dosage of ASPN-1, the damage to the tumor tissue was more obvious, and the cell gap was more obvious. Morphological observations showed that ASPN-1 may inhibit tumor cell proliferation by regulating the host immune response.

[0098] (4) Effect of ASPN-1 on serum cytokines in tumor-bearing mice

[0099] Peripheral blood was collected from mice and centrifuged at 4000 r / min for 15 min at 4°C to prepare serum. Serum concentrations of TNF-α, IL-2, and IFN-γ were detected using ELISA kits according to the manufacturer's instructions. All measurements were performed using a FlexA-200 enzyme-linked immunosorbent assay analyzer (Allsheng, China).

[0100] like Fig.14As shown in the figure, compared with the blank group, the levels of anti-tumor related cytokines (IL-2, TNF-α and IFN-γ) in the model group mice were significantly decreased (p<0.05). Compared with the model group, the levels of TNF-α, IL-2 and IFN-γ were significantly increased after ASPN-1 administration (p<0.05) in a dose-dependent manner. The cytokines in the 5-FU group were significantly reduced compared with the model group. The results showed that ASPN-1 may enhance immune function and inhibit tumor progression by promoting the release of cytokines in the anti-tumor response of tumor-bearing mice.

[0101] A CT26.WT tumor-bearing mouse model was constructed to evaluate the anti-cancer effect and mechanism of ASPN-1 in vivo. After oral treatment with ASPN-1, the volume and weight of mouse tumors decreased significantly, and the tumor growth inhibition rate was as high as 52.78%. At the same time, combined with the organ index, it can be found that ASPN-1 can protect the immune organs of tumor-bearing mice and improve spleen swelling and thymus atrophy. In addition, ASPN-1 can also increase the levels of tumor-related cytokines (IL-2, TNF-α and IFN-γ). It can be seen that ASPN-1 can inhibit the growth of tumors in vivo by regulating the body's immune ability.

Claims

1. A uniform Acanthopanax senticosus water-soluble glucan, characterized in that: The homogeneous Acanthopanax senticosus water-soluble glucan is composed of Glcp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→, →3,4)-Glcp-(1→, →4,6)-Glcp-(1→ residues in a molar ratio of 2.90:10.76:1.00:1.06:1.83; the main chain of the homogeneous Acanthopanax senticosus water-soluble glucan is composed of →4)-α-D-Glcp-(1→ residues; the weight average molecular weight Mw of the homogeneous Acanthopanax senticosus water-soluble glucan is 33.31 kDa.

2. A method for preparing uniform Acanthopanax senticosus water-soluble glucan, characterized in that: The steps include: S1: dissolving the ethanol precipitate obtained in the process of Acanthopanax senticosus injection in water, adding ethanol to the precipitate aqueous solution for re-precipitation, re-dissolving the precipitate for re-precipitation, removing protein by Sevage method, removing small molecule impurities by dialysis, and freeze-drying to obtain Acanthopanax senticosus crude polysaccharide; S2: Take crude polysaccharide of Acanthopanax senticosus, add water to make a solution with a mass percentage of 2-6%, load it onto a DEAE-52 cellulose column, elute with ultrapure water, and the elution rate is 1-5 mL / min. The eluted polysaccharide solution is concentrated and dialyzed at 4°C with a molecular weight cutoff of 3.5 kDa. The retained solution is freeze-dried, and the eluted product is taken as the separated sugar of Acanthopanax senticosus; S3: Take the Acanthopanax senticosus separated sugar, dissolve it in water, load it onto a Sephadex G-100 gel column, elute with ultrapure water at a rate of 0.5 to 1.5 mL / min, collect the main components according to the elution curve, dialyze at 4° C., use a molecular weight cutoff of 3.5 kDa, and freeze-dry to obtain the homogeneous Acanthopanax senticosus water-soluble glucan.

3. The method for preparing a uniform Acanthopanax senticosus water-soluble glucan according to claim 2, characterized in that: After the first alcohol precipitation in step S1, the precipitate is dissolved in water at a solid-liquid ratio of 1:160, the precipitate aqueous solution is concentrated under reduced pressure to 1 / 5 of the original volume, ethanol is added to make the ethanol concentration in the solution reach 75-85%, and the solution is allowed to stand at 4°C overnight and freeze-dried to obtain the Acanthopanax senticosus crude polysaccharide alcohol precipitate.

4. The method for preparing a uniform Acanthopanax senticosus water-soluble glucan according to claim 3, characterized in that: The method for removing protein by Sevage method in step S1 comprises the following steps: preparing the Acanthopanax senticosus crude polysaccharide alcohol precipitate with water to prepare an Acanthopanax senticosus crude polysaccharide alcohol precipitate aqueous solution with a concentration of 20 mg / mL; preparing chloroform and n-butanol into a mixed organic reagent in a volume ratio of 4:1, mixing the mixed organic reagent with the Acanthopanax senticosus crude polysaccharide alcohol precipitate aqueous solution in a volume ratio of 1:1, shaking for 30 minutes, centrifuging the mixed solution at 4000 rpm for 15 minutes, and collecting the uppermost sugar solution.

5. The method for preparing a uniform Acanthopanax senticosus water-soluble glucan according to claim 4, characterized in that: In the deproteinization step of the Sevage method, the sugar solution is further deproteinized using a mixed organic reagent of chloroform and n-butanol, and the deproteinization is performed 9 times in total.

6. The method for preparing a uniform Acanthopanax senticosus water-soluble glucan according to claim 2, characterized in that: In step S2, the dialysis time is 48 hours, and the distilled water is changed every 2 to 3 hours.

7. The method for preparing a uniform Acanthopanax senticosus water-soluble glucan according to claim 2, characterized in that: In step S3, the dialysis time is 48 hours, and the distilled water is changed every 2 to 3 hours.

8. Use of the homogeneous Acanthopanax senticosus water-soluble glucan according to claim 1 or the homogeneous Acanthopanax senticosus water-soluble glucan prepared by the preparation method of any one of claims 2 to 7 in the preparation of an immunopotentiator.

9. The use according to claim 8, characterized in that The immunopotentiator is an anti-tumor immunopotentiator.

10. The use according to claim 9, characterized in that: The tumor is colorectal cancer.

11. The use according to claim 8, characterized in that: The immunopotentiator is an oral preparation.

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