Astragalus polysaccharide APSN and its preparation method and application

Abstract

The invention discloses an astragalus polysaccharide APSN, comprising 1,4-linked α-D-Glcp, t-β-D-Glcp and 1,6-linked α-D-Glcp; the main chain skeleton is composed of 1,4-linked α-D-Glcp, the side chain is 1,6-linked α-D-Glcp and t-β-D-Glcp, the 1,6-linked α-D-Glcp is connected to any O-6 position of the 1,4-linked α-D-Glcp to form a 1,4,6-linked α-D-Glcp, and the t-β-D-Glcp is connected to the 1,6-linked α-D-Glcp. The astragalus polysaccharide APSN of the invention can induce RAW264.7 cells to transform from M1 subtype to M0 subtype, thereby reducing the generation of inflammation, and can also reduce the inflammatory response of HUVECs and promote the growth of HUVECs.

Description

Technical Field

[0001] The invention relates to the field of polysaccharides, in particular to astragalus polysaccharide APSN and a preparation method and application thereof. Background Art

[0002] Polysaccharides have become a hot topic of research because of their potential beneficial effects on human health. For example, polysaccharides from plants and fungi have strong immunomodulatory activity. Whether polysaccharides have immunological activity or not is usually related to their structure, such as monosaccharide, glycosidic bond composition, conformation, molecular weight, functional groups and branching characteristics.

[0003] Astragalus (Radix Astragalus) is a perennial herbaceous plant of the Leguminosae family, also known as Astragalus membranaceus. It is produced in Inner Mongolia, Shanxi, Gansu, Heilongjiang and other places, including Mongolian Astragalus and Membranous Astragalus. The Compendium of Materia Medica records that "Astragalus is the best for the elderly, and its color is yellow, so it is named as the best for the tonic". The medicinal use of Astragalus has a history of more than 2,000 years, and Astragalus polysaccharide is one of the main components of Astragalus.

[0004] Astragalus polysaccharide itself is a mixture with a relatively complex structure. Natural astragalus polysaccharide has a high molecular weight, with an average molecular weight of more than 100,000, and cannot be normally digested and absorbed by the human body. The main components of astragalus polysaccharides on the market are glucans and heteropolysaccharides, among which glucans include water-soluble and water-insoluble glucans, namely α-(1→4)(1→6) glucans and α-(1→4) glucans. Heteropolysaccharides are mostly water-soluble acidic heteropolysaccharides, mainly composed of glucose, rhamnose, arabinose and galactose. A small amount contains uronic acid, composed of galacturonic acid and glucuronic acid; while some heteropolysaccharides are composed only of glucose and arabinose. However, the relative molecular mass and volume of astragalus polysaccharides are generally large, and they are easy to form complexes with high viscosity and low solubility, which limits their diffusion and absorption in the body and affects their clinical application effect. Currently, there is little information on the isolation and purification of astragalus polysaccharides, and there is little research on the preparation of the active ingredients of astragalus polysaccharides. Many structural characteristics and pharmacological effects of the polysaccharides purified from astragalus polysaccharides have not yet been clarified, and there is no research on the immune activity of pure astragalus polysaccharides. Summary of the invention

[0005] Based on this, it is necessary to provide an astragalus polysaccharide APSN.

[0006] The first aspect of the present invention provides an astragalus polysaccharide APSN, comprising 1,4-linked α-D-Glcp, t-β-D-Glcp and 1,6-linked α-D-Glcp; the main chain skeleton is composed of 1,4-linked α-D-Glcp, and the side chains are 1,6-linked α-D-Glcp and t-β-D-Glcp, the 1,6-linked α-D-Glcp is connected to any O-6 position of the 1,4-linked α-D-Glcp to form a 1,4,6-linked α-D-Glcp, and the t-β-D-Glcp is connected to the 1,6-linked α-D-Glcp.

[0007] In one embodiment, in the astragalus polysaccharide APSN, the content of 1,4-α-D-Glcp is 54.4wt%, the content of 1,6-α-D-Glcp is 11.1wt%, the content of 1,4,6-α-D-Glcp is 14wt%, and the content of t-β-D-Glcp is 20.4wt%.

[0008] In one embodiment, the relative molecular weight of the astragalus polysaccharide APSN is 4970Da.

[0009] In one embodiment, the chemical composition of the astragalus polysaccharide APSN includes:

[0010] Total sugar content 98% to 99%;

[0011] Protein content: 1% to 2%.

[0012] In one embodiment, the astragalus polysaccharide APSN does not contain uronic acid.

[0013] In one embodiment, the branching degree of the astragalus polysaccharide APSN is 0.3-0.4.

[0014] The second aspect of the present invention provides a method for preparing astragalus polysaccharide APSN, comprising the following steps:

[0015] The crude astragalus polysaccharide is dissolved in 10 to 30 times the mass of water, and small molecules are removed by dialysis to obtain the crude astragalus polysaccharide with small molecules removed; the crude astragalus polysaccharide with small molecules removed is centrifuged to remove impurities; the supernatant after centrifugation is taken and eluted with a mobile phase and an anion exchange chromatography column, wherein the mobile phase is water, and the eluate is collected to prepare the astragalus polysaccharide APSN.

[0016] In one embodiment, the molecular weight cut-off for dialysis is 3400Da to 3500Da.

[0017] In one embodiment, the anion exchange chromatography column is a Toyopearl DEAE 650M column.

[0018] The third aspect of the present invention provides a use of the astragalus polysaccharide APSN in the preparation of a drug for inhibiting inflammatory response or a drug for promoting angiogenesis.

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

[0020] The physicochemical properties of the Astragalus polysaccharide APSN of the present application, including molecular weight and distribution, conformation, composition, polysaccharide content, protein content, chemical structure and anti-immune effect, were studied. The Astragalus polysaccharide APSN of the present application has a total sugar content of 98.8%, no uronic acid, a small amount of protein, high purity, and only contains glucose, which is a six-carbon aldose with a high degree of branching. At the same time, pharmacological experiments have found that the Astragalus polysaccharide APSN of the present invention can induce RAW264.7 macrophages to transform from the M1 subtype to the M0 type, thereby reducing the occurrence of inflammation. At the same time, the Astragalus polysaccharide APSN of the present invention can reduce the inflammatory response of HUVECs and promote the growth of HUVECs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a gradient elution curve of Astragalus polysaccharide APSN in one embodiment;

[0022] Figure 2 The chemical composition of Astragalus polysaccharide APSN in one embodiment;

[0023] Figure 3 This is an infrared spectrum of Astragalus polysaccharide APSN in one embodiment;

[0024] Figure 4 This is the total ion current diagram of Astragalus polysaccharide APSN in one embodiment;

[0025] Figure 5 The gas chromatography-mass spectrometry analysis diagram of Astragalus polysaccharide APSN in one embodiment: (A) total ion current of PMAAs in Astragalus polysaccharide APSN (B) diagnostic fragments (C) glycosyl residue fragmentation rules;

[0026] Figure 6 Astragalus polysaccharide APSN in one embodiment 1 H NMR (A) and 13 C NMR(B) spectrum;

[0027] Figure 7 HSQC (A) and HMBC (B) spectra of Astragalus polysaccharide APSN in one embodiment;

[0028] Figure 8 This is a structural diagram of Astragalus polysaccharide APSN in one embodiment;

[0029] Fig. 9 This is a graph of RAW264.7 cell proliferation in one embodiment;

[0030] Fig.10 This is a RAW264.7 live and dead staining diagram in one embodiment;

[0031] Fig.11 This is a diagram of HUVECs cell proliferation in one embodiment;

[0032] Fig.12 This is a picture of HUVECs cell death and viability staining in one embodiment;

[0033] Fig.13 This is a diagram of qRT-PCR determination of RAW264.7 inflammatory gene in one embodiment;

[0034] Fig.14 This is a diagram of qRT-PCR determination of HUVECs inflammatory genes in one embodiment;

[0035] Fig.15 This is a graph showing the NO determination of RAW264.7 in one embodiment;

[0036] Fig.16 This is a diagram of qRT-PCR determination of HUVECs angiogenic factors in one embodiment. DETAILED DESCRIPTION

[0037] The Astragalus polysaccharide APSN of the present invention is further described in detail below in conjunction with specific examples. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0039] the term

[0040] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0041] In the present invention, "optionally", "optional" and "optional" refer to optional, that is, to any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present invention, % (w / w) and wt% both represent weight percentage.

[0044] The first aspect of the present invention provides an astragalus polysaccharide APSN, comprising 1,4-linked α-D-Glcp, t-β-D-Glcp and 1,6-linked α-D-Glcp; the main chain skeleton is composed of 1,4-linked α-D-Glcp, and the side chains are 1,6-linked α-D-Glcp and t-β-D-Glcp, the 1,6-linked α-D-Glcp is connected to any O-6 position of the 1,4-linked α-D-Glcp to form a 1,4,6-linked α-D-Glcp, and the t-β-D-Glcp is connected to the 1,6-linked α-D-Glcp.

[0045] Optionally, in the astragalus polysaccharide APSN, the content of 1,4-α-D-Glcp is 54.4wt%, the content of 1,6-α-D-Glcp is 11.1wt%, the content of 1,4,6-α-D-Glcp is 14wt%, and the content of t-β-D-Glcp is 20.4wt%.

[0046] Optionally, the relative molecular weight of the astragalus polysaccharide APSN is 4970Da.

[0047] Optionally, the chemical composition of the astragalus polysaccharide APSN includes:

[0048] Total sugar content 98% to 99%;

[0049] Protein content: 1% to 2%.

[0050] Optionally, the astragalus polysaccharide APSN does not contain uronic acid.

[0051] Optionally, the branching degree of the astragalus polysaccharide APSN is 0.3-0.4.

[0052] The second aspect of the present invention provides a method for preparing astragalus polysaccharide APSN, comprising the following steps:

[0053] The crude astragalus polysaccharide is dissolved in 10 to 30 times the mass of water, and small molecules are removed by dialysis to obtain the crude astragalus polysaccharide with small molecules removed; the crude astragalus polysaccharide with small molecules removed is centrifuged to remove impurities; the supernatant after centrifugation is taken and eluted with a mobile phase and an anion exchange chromatography column, wherein the mobile phase is water, and the eluate is collected to prepare the astragalus polysaccharide APSN.

[0054] Optionally, the molecular weight cut-off for dialysis is 3400Da to 3500Da.

[0055] Optionally, the anion exchange chromatography column is a Toyopearl DEAE 650M column.

[0056] The third aspect of the present invention provides a use of the astragalus polysaccharide APSN in the preparation of a drug for inhibiting inflammatory response or a drug for promoting angiogenesis.

[0057] The following is further described in conjunction with specific examples and comparative examples. The raw materials involved in the following specific examples, unless otherwise specified, can all be sourced from commercial sources. The instruments used, unless otherwise specified, can all be sourced from commercial sources. The processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0058] The following are specific embodiments.

[0059] Example 1

[0060] Isolation and structural identification of Astragalus polysaccharide APSN

[0061] 1 Isolation of Astragalus polysaccharide APSN

[0062] The purchased crude Astragalus polysaccharide APSC was dissolved by adding 20 times the mass of pure water, fully stirred at room temperature of 37°C, and the sample was placed in a dialysis bag with a molecular weight of 3500Da. After dialysis with pure water for 3 days, the solution in the bag was collected, concentrated and freeze-dried to obtain crude Astragalus polysaccharide (APSP) with small molecules removed. The crude polysaccharide treated above was added with 20 times the mass of pure water to completely dissolve it, and then centrifuged 3 times at 5000r / min for 20 minutes to remove water-insoluble impurities. The supernatant was taken and passed through a fully loaded and balanced anion exchange chromatography column Toyopearl DEAE 650M column (5.0cm×20cm), and eluted with H2O, 0.5M and 1M NaCl as eluents in turn to separate neutral polysaccharides and acidic polysaccharides. The eluate was detected at 490nm by the phenol-sulfuric acid method, and the partial eluate containing polysaccharides was collected in sections of 20mL per tube until no polysaccharides were detected. The gradient elution curve is shown as follows Figure 1As shown in the figure, the neutral polysaccharide in the H2O elution part has a high content, which is named astragalus polysaccharide APSN (yield: 36% of APSC). Astragalus polysaccharide APSN is concentrated, freeze-dried, and stored in a desiccator.

[0063] 2 Analysis of structural characteristics of Astragalus polysaccharide APSN

[0064] 2.1 Molecular weight distribution

[0065] The molecular weight distribution of Astragalus polysaccharide APSN was determined by high performance liquid chromatography (HPGPC). The standard used for molecular weight detection was Dextrans (300-5×106 g / mol).

[0066] (1) Preparation of standard solution and drawing of standard curve: Weigh 5 mg of Dextrans standard of different molecular weights respectively, dissolve in pure water to prepare 5 mg / mL solution, filter with 0.45 μm filter membrane for liquid chromatography detection. Draw a linear regression equation with retention time as x variable and logarithm of molecular weight as y variable.

[0067] (2) Preparation and determination of sample solution: Weigh 5 mg of Astragalus polysaccharide APSN sample and dissolve it in pure water to prepare a 5 mg / mL solution, and filter it using a 0.45 μm filter membrane for liquid chromatography detection. Substitute the obtained characteristic peak retention time into the standard curve to calculate the molecular weight distribution of the Astragalus polysaccharide APSN sample.

[0068] (3) Chromatographic conditions: Elite P230 chromatograph (Elite, Dalian) equipped with an evaporative light scattering detector; the chromatographic column was PL aquagel-OH MIXED-H (7.5 mm × 300 mm, 8 μm); the mobile phase was 0.1 M NaNO3, the flow rate was 0.6 mL / min, and the column temperature was controlled at 40°C.

[0069] (4) Test results: Figure 2 As shown, the results show that the Astragalus polysaccharide APSN sample presents a uniform and symmetrical peak, indicating that the Astragalus polysaccharide APSN has good uniformity. The peak areas are respectively substituted into the standard curve, and the apparent molecular weight of Astragalus polysaccharide APSN is 4970Da.

[0070] 2.2 Analysis of chemical composition of Astragalus polysaccharide APSN

[0071] 2.2.1 Determination of total sugar content

[0072] The total sugar content of Astragalus polysaccharide APSN was determined by phenol-sulfuric acid method. The standard substance used for total sugar content was galactose (Gal).

[0073] (1) Preparation of standard solution and drawing of standard curve: Weigh 5 mg of galactose standard accurately, dissolve it in pure water and prepare a 1 mg / mL stock solution. Use a pipette to draw the glucose stock solution to gradually dilute the series of standard solutions. Use a pipette to draw 200 μL of each standard solution of different concentrations, add 0.2 mL of 5% phenol solution, and then quickly add 1 mL of concentrated sulfuric acid, shake and mix, and react for 20 minutes. Use pure water (0 μg / mL) as the blank control group, and measure its absorbance A at a wavelength of 490 nm. Use glucose concentration C as the x variable and absorbance A as the y variable to make a linear regression equation. The linear regression equation is y=0.0684x-0.0796.

[0074] (2) Preparation and determination of sample solution: Weigh 1 mg of Astragalus polysaccharide APSN accurately, add pure water to fully dissolve, and prepare a 1 mg / mL sample solution. Use a pipette to take 200 μL of each sample solution, add 1800 μL of pure water to dilute it into a 100 μg / mL sample solution. Use a pipette to take 200 μL of each sample solution, add 0.2 mL of 5% phenol solution, then add 1 mL of concentrated sulfuric acid, shake and mix, react for 20 minutes, and measure its absorbance A at a wavelength of 490 nm. Substitute it into the standard curve to calculate the total sugar content of Astragalus polysaccharide APSN.

[0075] 2.2.2 Determination of uronic acid content

[0076] The m-hydroxybiphenyl method was used to determine the uronic acid content of APSN. The standard substance used for the uronic acid content determination was glucuronic acid (GlcA).

[0077] (1) Preparation of standard solution and drawing of standard curve: Weigh 5 mg of GlcA standard accurately, add pure water to fully dissolve, and prepare a 1 mg / mL standard stock solution. Use the standard stock solution and dilute it to 1 mL with pure water to prepare 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL standard solutions. Use a pipette to draw 200 μL of each of the 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL standard solutions, add 1.2 mL of 0.0125 M tetraborate-concentrated sulfuric acid solution, cool the test tube in ice to room temperature, and heat it in a boiling water bath for 5 min. After heating, place it in ice to cool to room temperature, and add 20 μL of 0.15% m-hydroxybiphenyl solution. A blank group was set up at each concentration, except that 20 μL of 0.15% m-hydroxybiphenyl solution was replaced by 20 μL of 0.5% NaOH solution. The absorbance of each group of standard solutions was measured at a wavelength of 520 nm after 5 minutes of reaction. The absorbance A obtained after deducting the absorbance of the blank group was used as the y variable, and the galacturonic acid concentration C was used as the x variable to make a linear regression equation. The linear regression equation was y=0.0684x-0.0796.

[0078] (2) Preparation and determination of sample solution: Weigh 1 mg of Astragalus polysaccharide APSN sample accurately, add an equal amount of pure water to fully dissolve, and prepare a 1 mg / mL sample stock solution. Use a pipette to draw 50 μL of the sample stock solution, add 950 μL of pure water to fully dissolve, and dilute to a 50 μg / mL sample solution. Use a pipette to draw 200 μL of the sample solution respectively, cool the test tube in ice to room temperature, and then heat it in a boiling water bath for 5 minutes. After heating, place it in ice to cool to room temperature, and add 20 μL of 0.15% m-hydroxybiphenyl solution. Set up a blank group. The treatment of the blank group is the same as before, except that the 20 μL 0.15% m-hydroxybiphenyl solution is replaced by 20 μL 0.5% NaOH solution. The absorbance of the Astragalus polysaccharide APSN sample solution was measured at a wavelength of 520 nm after the reaction for 5 minutes, and the absorbance A obtained after deducting the absorbance of the blank group was substituted into the standard curve to calculate the uronic acid content of the Astragalus polysaccharide APSN sample.

[0079] 2.2.3 Determination of protein content of Astragalus polysaccharide APSN

[0080] The protein content of Astragalus polysaccharide APSN was determined using the Bio-Time protein quantification kit. The standard used for protein content determination was bovine serum albumin (BSA).

[0081] (1) Preparation of standard solution and drawing of standard curve: Use a pipette to draw 5 mg / mL bovine serum albumin standard and dilute it with water to prepare solutions of 0 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, 1500 μg / mL, and 2000 μg / mL. Use a pipette to draw 100 μL of each concentration of standard solution and add it to a 96-well plate, then add 100 μL of Bio-rad protein analysis kit solution, set three replicates for each concentration, mix and react on an oscillator for 5 minutes, and measure the absorbance at a wavelength of 562 nm on an enzyme reader. Draw a linear regression equation with BSA concentration C as the x variable and absorbance A as the y variable. The linear regression equation is y=1.250x+0.036.

[0082] (2) Preparation and determination of sample solution: Weigh 5 mg of Astragalus polysaccharide APSN accurately, add pure water to fully dissolve, and prepare a 1 mg / mL sample stock solution. Use a pipette to draw the Astragalus polysaccharide APSN sample stock solution, add pure water to dilute, and prepare 100 μg / mL and 50 μg / mL Astragalus polysaccharide APSN sample solutions. Use a pipette to draw 100 μL of each sample solution and add it to a 96-well plate, then add 100 μL of Bio-rad protein analysis kit solution. Set up three replicates for each sample, mix and react on an oscillator for 5 minutes, and measure the absorbance at a wavelength of 562 nm on an ELISA reader. Substitute the absorbance of each sample into the standard curve to calculate the protein content of Astragalus polysaccharide APSN.

[0083] 2.2.4 Determination of monosaccharide composition

[0084] (1) Uric acid hydrolysis: 20 mg of Astragalus polysaccharide APSN sample was accurately weighed into a stoppered reaction tube, 10 mL of 1 M imidazole-hydrochloric acid buffer (pH 7.0) was added, and the mixture was stirred in an ice bath for 30 min; then 200 mg of sodium borohydride was slowly added every 5 min, and stirred after adding three times until no bubbles were generated; 10% acetic acid / methanol solution was added to remove excess sodium borohydride, and the mixture was stirred until the solution was clear and no bubbles were generated; and dialyzed for 40 h.

[0085] (2) Hydroxyl reduction: The dialysate was concentrated to about 10 mL, and the pH was adjusted to 4.75 with 0.5% NaOH and 0.2N HCl; 250 mg of carboxymethyl cellulose was slowly added to maintain the pH at 4.75 for 2 h; the solution was cooled in ice, 2.5 mL of 4 M imidazole-hydrochloric acid buffer (pH 7.0) was added while stirring, and 200 mg of sodium borohydride was slowly added twice, each time with an interval of 5 min, and stirred in an ice bath for 1 h; 10% acetic acid / methanol solution was added to remove excess sodium borohydride, and the solution was stirred until it was clear and no bubbles were generated; dialyzed for 40 h; concentrated by rotary evaporation, lyophilized, weighed, and the yield was calculated.

[0086] 2.2.5 Analysis results

[0087] The analysis results of total sugar content, protein content and uronic acid content of Astragalus polysaccharide APSN are shown in Table 1. The total sugar content of Astragalus polysaccharide APSN is 98.8%, without uronic acid, and with a small amount of protein, which shows that the purity of Astragalus polysaccharide APSN is very high and is almost entirely composed of polysaccharides.

[0088] Table 1

[0089] Chemical composition (%) Astragalus polysaccharide APSN Total sugar content 98.8 Protein Content 1.51 Uric acid content -

[0090] 2.3 Fourier transform infrared spectroscopy FT-IR analysis of Astragalus polysaccharide APSN

[0091] (1) Analysis method: Take 2 mg of dried Astragalus polysaccharide APSN sample, add 200 mg KBr, grind and mix in an agate mortar, press into tablets, and perform infrared scanning in the range of 4000-400 cm-1 using an infrared spectrometer.

[0092] (2) Analysis results: The infrared spectrum analysis results are as follows Figure 3 As shown, the characteristic absorption peak at 3373cm-1 is the OH stretching vibration of the sugar residue. The absorption peak at 2928.8cm-1 is the CH stretching vibration on the sugar ring. The absorption signal near 1617cm-1 is attributed to the bending vibration of HOH. The peaks at 1358cm-1 and 1396cm-1 are the bending vibrations of the CH bond. In addition, the two strong peaks in the range of 1100 to 1000cm-1 are the stretching vibration peaks of the pyranose ring. The absorption peak at 847cm-1 is for the α-configuration sugar ring. No characteristic absorption peaks of uronic acid and sulfate groups were found, proving that Astragalus polysaccharide APSN is a neutral polysaccharide.

[0093] 2.4 Preparation of acetylated products

[0094] (1) Acid hydrolysis: Accurately weigh the astragalus polysaccharide APSN before and after reduction into a stoppered reaction tube, add 1 mL of freshly prepared 2 M trifluoroacetic acid (TFA) solution using a pipette, heat in a high-temperature electric heating reactor at 120 °C for 2 h, and cool to room temperature after heating. Dry the solvent with nitrogen, and add methanol to help evaporate the water in the solution as quickly as possible.

[0095] (2) Reduction: Add 1 mL of 1 M ammonia water and 3 mg of sodium borohydride to the stoppered reaction tube after acid hydrolysis, and react overnight at room temperature; add 10% acetic acid / methanol solution to remove excess sodium borohydride, use a rotary evaporator to remove the acid, repeat adding 10% acetic acid / methanol solution and rotary evaporation three times, then add methanol and rotary evaporation four times, until completely dry, scrape off the powder on the wall of the collection bottle.

[0096] (3) Acetylation: 1 mL of acetic anhydride and 100 μL of 1-methylimidazole were added to the reduced 10 mL rotary evaporator, shaken to make them fully contact, and placed at room temperature for 30 min; 1 mL of pure water was added to each sample in ice to terminate the reaction, and the solution was transferred to a stoppered reaction tube; extracted with chloroform twice, and the chloroform layer was collected and combined; extracted with pure water, centrifuged at 3500 r / min for 3 min, and then the water layer was discarded, and the chloroform layer was retained. This operation was repeated 5 times; anhydrous sodium sulfate was added to each stoppered reaction tube, and placed at room temperature for 30 min. The solution in the tube was aspirated with a rubber-tipped dropper, and 1 mL of chloroform was added to wash the remaining material. The washing solution was combined with the sample solution, and the sample solution was filtered with a small column filled with anhydrous sodium sulfate to fully remove the water in the solution; the solvent was blown dry with nitrogen; 1 mL of chloroform was added and shaken to mix well to obtain the chloroform solution of Astragalus polysaccharide APSN for GC-MS determination.

[0097] (4) Preparation of standard solution: Nine kinds of precise monosaccharide standards (Rha, Fuc, Ara, Xyl, Man, Glc, Gal) were treated with acid hydrolysis, reduction, and acetylation according to the steps (1) to (3), and then 1 mL of chloroform was added and mixed by vortexing to obtain a chloroform solution of the standard for use in GC-MS determination.

[0098] (5) GC-MS determination:

[0099] aAnalysis conditions: Instrument: Agilent 7890A-5975C gas chromatograph-mass spectrometer; Chromatographic column: ZB-5MScolumn (30m×0.25mm id, 0.10μm, Phenomenex, USA); Chromatographic conditions: Inlet temperature: 240℃; Split ratio: 20:1; Program temperature: 170℃~190℃, Heating rate: 1℃ / min; Final temperature of 240℃ was maintained for 5min; Carrier gas: nitrogen.

[0100] b Analysis method: The chloroform solution of the standard and the chloroform solution of Astragalus polysaccharide APSN are subjected to GC-MS determination and analysis to obtain the corresponding total ion flow chart, and the characteristic cleavage fragments of the sample solution are compared with the standard spectrum to obtain the monosaccharide composition results of Astragalus polysaccharide APSN.

[0101] (6) Analysis results: Total ion current (TIC) results are as follows Figure 4 As shown, Figure 4 (A) is the chromatogram of the mixed monosaccharide standard, from left to right: rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose. Figure 4 (B) is the TIC graph of the sample. It can be seen from the figure that Astragalus polysaccharide APSN only contains glucose, which is a six-carbon aldose.

[0102] 2.5 Analysis of sugar chain connection

[0103] (1) Analytical method: Take 1 mg of dried Astragalus polysaccharide APSN sample in a 2 mL reaction bottle and place it in a desiccator overnight. Add 1 mL of DMSO and stir until the sample is completely dissolved. Grind 50 mg of NaOH into fine powder in a mortar, add it to the reaction bottle, and stir for 2 h. Add 150 μL of iodomethane every 15 min for a total of 3 times and stir at room temperature for 45 min until the solution is clear and transparent. Add 1 mL of pure water to terminate the reaction, put it in an ice bath, transfer it to a glass reaction tube, blow dry the iodomethane with N2, extract it with an equal amount of chloroform twice, combine the chloroform layers, wash it with pure water 5 times, and discard the water layer. Blow dry the solvent in the chloroform layer with N2, add 0.5 mL of 2 mol / L TFA, hydrolyze it at 120 °C for 2 h, let it stand to room temperature, blow dry the solvent with N2, and wash it with methanol 3 times. Add 0.5 mL of 1 mol / L ammonia solution, then add 3 mg of sodium borodeuteride powder, tighten the reaction tube, and leave it at room temperature overnight. Add 10% volume fraction of acetic acid-methanol solution, spin dry the solvent with a rotary evaporator, and repeat the operation 3 times. Add anhydrous methanol to dissolve again, spin dry the solvent, and repeat the operation 4 times. Add 1mL of acetic anhydride and 0.1mL of 1-methylimidazole, and carry out acetylation reaction at room temperature for 30 minutes. Transfer the solution to a reaction tube, add 1mL of pure water to terminate the reaction, and ice bath. Add 1mL of chloroform to extract twice, collect the chloroform layer, combine and transfer to a clean reaction tube, add 2mL of pure water to wash 5 times, and discard the water layer. Add anhydrous sodium sulfate to remove water from the solution. After standing for 30 minutes, pass the solution through a small column of anhydrous sodium sulfate, collect it, blow it to 0.5mL with N2, and set it aside.

[0104] (2) Analytical conditions: Instrument: Agilent 7890A-5975C gas chromatograph-mass spectrometer; Chromatographic column: ZB-5MScolumn (30 m×0.25 mm id, 0.10 μm, Phenomenex, USA); Chromatographic conditions: Inlet temperature: 120°C; Split ratio: 20:1; Program temperature: 120°C to 280°C, heating rate: 2°C / min; 190°C to 240°C, heating rate: 5°C / min; Finally, keep at 240°C for 5 min; Carrier gas: nitrogen.

[0105] (3) Analysis results: According to the retention time of the Astragalus polysaccharide APSN sample, the mass spectrum of the sample's fragment molecules was determined. The total particle flow diagram TIC diagram results of the mass spectrum are as follows: Figure 5 shown.

[0106] From the mass spectrum data of peak 1, Figure 5As shown in (B), from the almost similar amounts of m / z 161 and 162, it can be seen that there should be two adjacent CC bonds connected by -OMe at C-3 and C-4 (with the same R group), and the secondary cleavage of m / z 162 is m / z 102, that is, the cleavage of one -CH3COOH. From m / z 118 and 205, it can be seen that there are also cleavages of adjacent CC bonds connected by -OMe at C-2 and C-3, so it can be judged that the secondary cleavage of m / z 162 is the cleavage of -OAc connected to C-3. The secondary cleavage of m / z 161 is m / z 101 and 129, which can be judged that C-5 is substituted by -OAc and C-6 is substituted by -OMe. Peak 1 is 1,5-diacetoxy-(1-deuterium)-2,3,4,6-tetramethoxyglucitol, which is the signal peak of terminal glucose, that is, t-Glc. From the mass spectrometry data of peak 2, we can see that the m / z 118 and 233 with the highest content represent two adjacent -OMe at C-2 and C-3. The secondary fragmentation of m / z 233 is m / z 173 and 113, which shows that the -OMe on C-5 is replaced by -OAc. However, since there is no m / z 161, the -OMe on C-4 is replaced by -OAc, and C-6 is replaced by -OMe. Peak 2 is 1,4,5-triacetoxy-(1-deuterium)-2,3,6-trimethoxyglucitol, which is the signal peak of 1,4-linked glucose. From the mass spectrometry data of peak 3, we can see that the characteristic peaks of m / z 162 and 189 represent two adjacent -OMe at C-3 and C-4. The secondary fragmentation of m / z 189 is m / z 129, which shows that the -OMe on C-6 is replaced by -OAc. Peak 3 is 1,5,6-triacetoxy-(1-deuterium)-2,3,4-trimethoxyglucitol, which is the signal peak of 1,6-linked glucose. From the mass spectrum data of peak 4, it can be seen that the characteristic peaks of m / z 118 and 261 represent two adjacent -OMe at C-2 and C-3. It can be inferred that the -OMe on C-4, C-5 and C-6 of peak 4 is replaced by -OAc. Peak 4 is 1,4,5,6-tetraacetoxy-(1-deuterium)-2,3-dimethoxyglucitol, which is the signal peak of 1,4,6-linked glucose.

[0107] In summary, peak 1 is 1,5-diacetoxy-(1-deuterium)-2,3,4,6-tetramethoxyglucitol, which is the signal peak of terminal glucose, i.e. t-Glc, peak 2 is 1,4,5-triacetoxy-(1-deuterium)-2,3,6-trimethoxyglucitol, which is the signal peak of 1,4-linked glucose, peak 3 is 1,5,6-triacetoxy-(1-deuterium)-2,3,4-trimethoxyglucitol, which is the signal peak of 1,6-linked glucose, and peak 4 is 1,4,5,6-tetraacetoxy-(1-deuterium)-2,3-dimethoxyglucitol, which is the signal peak of 1,4,6-linked glucose.

[0108] (4) Calculation of fragment molecular content: The relative content of Astragalus polysaccharide APSN was calculated based on the peak area ratio of peak 1, peak 2, peak 3, and peak 4. The results are shown in Table 2. It can be seen that Astragalus polysaccharide APSN is mainly composed of 1,4-Glcp (54.4%) as the main chain and has a branch 1,4,6-Glcp (14.0%) at the C-6 position. A Glcp is connected with 1,6 at each branch point. From the high performance gel permeation chromatography HPGPC ( Figure 1 ) The results show that there are a large number of oligosaccharides in Astragalus polysaccharide APSN, which may be easily depolymerized into monosaccharides during the methylation process, so there is a large amount of t-Glcp (20.4%).

[0109] Table 2. Linkage types and ratios of polysaccharides

[0110]

[0111] (5) Calculation of branching degree: The branching degree in Astragalus polysaccharide APSN was calculated from the molar ratio of sugar residues in Table 2, using the formula DB = (NT + NB) / (NT + NB + NL). DB is the branching degree, NT is the number of terminal sugars, NB is the number of branched sugars, and NL is the number of linear sugars. The NT, NB and NL of Astragalus polysaccharide APSN are 20.4%, 14.0% and 54.4%, respectively. Substituting into the formula, DB is 0.387. From the above results, it can be seen that Astragalus polysaccharide APSN has a high branching degree.

[0112] 2.6 Nuclear magnetic resonance analysis

[0113] (1) Analytical method: APSN was dried in vacuum on P2O5 for 72 h and then deuterium exchanged by freeze drying using D2O (0.5 mL) as solvent. Nuclear magnetic resonance spectroscopy was performed using a Bruker Avance 400 spectrometer. l H-NMR, 13 C-NMR, HSQC and HMBC spectral analysis. 1 H chemical shifts were referenced to HDO at δ 4.79 ppm as an internal standard.

[0114] (2) Analysis results:

[0115] exist 1 H-NMR spectrum ( Figure 6In A), the chemical shift signals are concentrated in a narrow region of 3.0ppm to 5.3ppm, where the terminal protons are considered to be in the region of 4.5ppm to 5.5ppm. Three strong signal peaks appear in this region, indicating that there are three different monosaccharide hydrogen atoms in the terminal hydrogen. In addition, the proton chemical shifts occurring in the region of 4.1ppm to 3.0ppm are assigned to other sugar residues.

[0116] exist 13 C-NMR spectrum ( Figure 6 In B), the major ectopic carbon signal is mainly observed between δ97 and δ110, and further indicates the presence of α- and β-glycosidic residues.

[0117] In the HSQC spectrum ( Figure 7 In A), the chemical shifts of all residues were elucidated in the HSQC spectra and the results are summarized in Table 3.

[0118] according to 1 H-NMR, 13 C-NMR, HSQC spectra, and methylation results suggest that three other anomeric signal assignments at δ99.66 / 5.40, δ98.56 / 4.56, and δ99.82 / 5.35 were identified as 1,4-, 1,6-, and 1,4,6-linked α-C1 / H1α-D-Glcp residues (B, C, and D). The low-shift chemical shifts at C-4 and C-6 suggest that residues B, C, and D are substituted at O-4, O-6, and O-4 / 6, respectively.

[0119] Table 3

[0120]

[0121] Inter-residue and intra-residue linkage sequences are inferred from HMBC spectra, such as Figure 7 B. Strong cross peaks (δ99.66 / 3.65 and δ76.73 / 5.40) show correlations between C-1 / H-4 and C-4 / H-1 of internal residue B, indicating the presence of a 1,4-linked α-glucan backbone. Although there is overlap in the cross peaks (δ99.66 / 3.65 and δ76.73 / 5.40), it also shows correlations between C-1 of residue D and H-4 of residue B with C-4 of residue D and H-1 of residue B, further indicating that residues B and D are associated as B-(1→4)-D. The cross peak (δ69.35 / 4.96) shows correlations between C-6 of residue D and H-1 of residue C, indicating an E-(1→6)-D linkage. Finally, the weak cross peak (δ 68.14 / 4.52) indicated a correlation between C-6 of residue C and H-1 of residue A, indicating that residues C and A were connected in the form of C-(1→6)-A.

[0122] Based on the results of HPGPC, monosaccharide composition, methylation, and NMR analysis, the structure of Astragalus polysaccharide APSN was fully elucidated. The main chain skeleton is composed of 1,4-linked α-D-Glcp, and the side chains are 1,6-linked α-D-Glcp and t-β-D-Glcp. The 1,6-linked α-D-Glcp is connected to any O-6 position of the 1,4-linked α-D-Glcp to form a 1,4,6-linked α-D-Glcp, and the t-β-D-Glcp is connected to the 1,6-linked α-D-Glcp.

[0123] These results suggest that APSN is a short-side-chain and highly branched glucan, and the hypothetical structure is Figure 8 shown.

[0124] 3 In vitro cell experiments

[0125] 3.1 Cell culture

[0126] RAW264.7 cells and human umbilical vein endothelial cells (RAW264.7 cells and HUVECs, obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured with Dulbecco's modified Eagle's medium (DMEM, high glucose, Gibco) supplemented with 5% or 10% fetal bovine serum and 1% penicillin-streptomycin, respectively, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0127] 3.2 Cell proliferation and live / dead staining

[0128] (1) RAW264.7 cell proliferation and live / dead staining

[0129] Experimental methods: RAW264.7 cells were co-cultured with APSN polysaccharide medium with different concentration gradients of APS (Normal group, APSN 25ug / mL group, APSN 50ug / mL group, APSN 250ug / mL group, APSN 500ug / mL group), and the viability and proliferation were marked as normal, APSN 25, APSN 50, APSN 250, and APSN 500, respectively, and evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) assay and live / dead staining kit, respectively. The cells were washed twice with PBS and then treated with a mixture of 10uL 1mmol / L calcein-AM reagent and 15uL13.5umol / L PI (Calcein-AM / PI double staining kit, DOJINDO, Japan) in 5mL PBS. After incubation at 37°C in the dark for 15 minutes, the cells were observed under an inverted fluorescence microscope (Leica).

[0130] Experimental results: The proliferation of RAW264.7 cells was detected by MTS method. Fig. 9 As shown, RAW264.7 cells gradually proliferated as the culture time increased. After the first day of culture, APSN 500μg / mL was the highest in each group, and the 250μg / mL dosing concentration group also showed a certain proliferation trend. On the third day of culture, compared with the normal group, each dosing concentration group also showed a certain dose-dependent proliferation trend, among which APSN 500μg / mL was the highest in each group. After the fifth day of culture, compared with each dosing concentration, there was obvious inhibition at the dosing concentration of 500μg / mL, but it was also able to promote cell proliferation compared with the normal group, among which the cell proliferation was most obvious at the dosing concentration of 250μg / mL. Live / dead cell graph ( Fig.10 ) It can be seen that the number of living cells at each administration concentration on the first, third, and fifth days was higher than that of the normal group, indicating that the administration of astragalus polysaccharide APSN can actively promote the proliferation of RAW264.7 cells.

[0131] (2) HUVECs cell proliferation and live / dead staining

[0132] Experimental methods: HUVECs cells were co-cultured with APSN polysaccharide medium with different concentration gradients of APS (Normal group, APSN 25ug / mL group, APSN 50ug / mL group, APSN 250ug / mL group, APSN 500ug / mL group), and the viability and proliferation were marked as normal, APSN 25, APSN 50, APSN 250, and APSN 500, respectively, and evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) assay and live / dead staining kit, respectively. The cells were washed twice with PBS and then treated with a mixture of 10uL 1mmol / L calcein-AM reagent and 15uL 13.5umol / LPI (Calcein-AM / PI double staining kit, DOJINDO, Japan) in 5mL PBS. After incubation at 37°C in the dark for 15 minutes, the cells were observed under an inverted fluorescence microscope (Leica).

[0133] Experimental results: The proliferation of HUVECs cells was detected by MTS method. Fig.11As shown, HUVECs cells gradually proliferate as the culture time increases. After the first day of culture, compared with the normal group, 250μg / mL was the highest in each group, and other dosing concentration groups also showed a certain proliferation trend. On the third day of culture, compared with the normal group, the proliferation trend of each dosing concentration group remained unchanged, and all were able to promote cell proliferation, among which 250μg / mL was the highest in each group. After the fifth day of culture, the cell proliferation was most obvious at the dosing concentration of 250μg / mL when compared with each dosing concentration. Live / dead cell diagram ( Fig.12 ) showed that the number of live cells at the fifth day in the 250μg / mL and 500μg / mL administration concentrations was significantly higher than that in the normal group, indicating that astragalus polysaccharide APSN can actively promote HUVECs cell proliferation within the administration range of 250μg / mL to 500μg / mL.

[0134] 3.3 Effects of Astragalus polysaccharide APSN on inflammatory factors

[0135] Experimental methods: To further study the effect of Astragalus polysaccharide APSN on inflammation, LPS was used to stimulate RAW264.7 cells and HUVECs cells to establish an in vitro inflammatory model. LPS can induce macrophages to secrete a large number of proinflammatory cytokines, such as iNOS, TNF-α, IL-23 and IL-1β. The inflammatory response stimulated by LPS can also promote the expression of related mRNA. The specific primers in Table 4 were used to detect the production of proinflammatory cytokines (iNOS, TNF-α, IL-1β, IL-23) and the anti-inflammatory cytokine IL-10 in LPS-stimulated cells and the expression levels of related mRNA by qRT-PCR.

[0136] Experimental results: Fig.13 As shown in the figure, the mRNA expression level of pro-inflammatory cytokines in LPS-induced RAW264.7 cells was significantly higher than that in the blank control group, confirming that LPS has successfully induced inflammation in RAW264.7 cells. APSN 250μg / mL can inhibit the mRNA expression of iNOS, TNF-α, IL-1β, IL-23 and increase IL-10 in LPS-induced RAW264.7 cells. Fig.14 As shown in the figure, the mRNA expression levels of proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-18 in LPS-induced HUVECs cells were significantly higher than those in the blank control group, and the difference was statistically significant (P<0.05), confirming that LPS has successfully induced inflammation in HUVECs. After 2 days of administration of APSN 250μg / mL, the mRNA expression of TNF-α, IL-1β, IL-6 and IL-18 in LPS-induced HUVECs cells was inhibited, and there were statistical differences (P<0.05).

[0137] The above results indicate that APSN 250 μg / mL can significantly downregulate the mRNA expression of inflammatory cytokines and thus reduce LPS-induced endothelial damage.

[0138] 3.4 Effect of Astragalus polysaccharide APSN on LPS-induced NO secretion in RAW264.7 macrophages

[0139] The effect of APS on NO production in LPS-induced RAW264.7 cells was evaluated by a nitric oxide detection kit (S00021S, Beyotime, China).

[0140] RAW264.7 cells were seeded in 96-well plates (2×105 cells / well) and cultured for 24 hours. The old culture medium was removed, and 100 μL of normal culture medium labeled Normal was added to one group, 100 μL of normal culture medium containing 0.5 μg / mL LPS was added to one group, and 100 μL of normal culture medium containing 0.5 μg / mL LPS and 250 μg / mL APSN was added to one group, labeled LPS-APSN 250. The three groups were cultured for 24 hours, and the NO concentration in the supernatant was detected using Griess reagent. Each group had 6 replicates. The absorbance of the reaction product was measured at 560 nm using an automatic microplate reader (Bio Red).

[0141] The results of the NO secretion test of Astragalus polysaccharide APSN, such as Fig.15 As shown in the results, compared with the blank control group, the NO content in the LPS positive group was increased, and the difference was statistically significant (P<0.05); Astragalus polysaccharide APSN could significantly downregulate the secretion of NO at a concentration of 250 μg / mL, and the difference was statistically significant (P<0.05), indicating that Astragalus polysaccharide APSN has anti-inflammatory activity.

[0142] 3.5 Effect of Astragalus polysaccharide APSN on angiogenic factors of HUVECs

[0143] VEGF, VEGFα, EGF and EGFL6 are among the most important angiogenesis regulators, which can induce endothelial cell proliferation and stimulate cell migration and invasion. The production of proinflammatory cytokines (VEGF, VEGFα, EGF and EGFL6) and the expression levels of related mRNA in LPS-stimulated cells were detected by qRT-PCR using specific primers in Table 4. Fig.16 As shown in the figure, after 2 days of administration of APSN 250 μg / mL, the mRNA expressions of VEGF, VEGFα, EGF and EGFL6 in HUVECs cells were promoted, which were statistically different from those in normal cells (P<0.05), indicating that 250 μg / mL Astragalus polysaccharide APSN has strong angiogenic activity.

[0144] Table 4

[0145]

[0146] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0147] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An astragalus polysaccharide APSN, characterized in that: It includes 1,4-linked α-D-Glcp, t-β-D-Glcp and 1,6-linked α-D-Glcp; the main chain skeleton is composed of 1,4-linked α-D-Glcp, the side chains are 1,6-linked α-D-Glcp and t-β-D-Glcp, the 1,6-linked α-D-Glcp is connected to any O-6 position of the 1,4-linked α-D-Glcp to form a 1,4,6-linked α-D-Glcp, and the t-β-D-Glcp is connected to the 1,6-linked α-D-Glcp; In the astragalus polysaccharide APSN, the content of 1,4-α-D-Glcp is 54.4wt%, the content of 1,6-α-D-Glcp is 11.1wt%, the content of 1,4,6-α-D-Glcp is 14wt%, and the content of t-β-D-Glcp is 20.4wt%; The relative molecular weight of the astragalus polysaccharide APSN is 4970Da; The chemical composition of the astragalus polysaccharide APSN includes: Total sugar content 98%~99%; Protein content: 1%~2%.

2. The astragalus polysaccharide APSN according to claim 1, characterized in that The astragalus polysaccharide APSN does not contain uronic acid.

3. The astragalus polysaccharide APSN according to claim 2, characterized in that The branching degree of the astragalus polysaccharide APSN is 0.3-0.

4.

4. Use of the astragalus polysaccharide APSN according to any one of claims 1 to 3 in the preparation of drugs for inhibiting inflammatory response or promoting angiogenesis.

Images