CN108359024B

CN108359024B - An ethanol soluble oligosaccharide containing radix astragali, and its preparation method and anti-tumor application

Info

Publication number: CN108359024B
Application number: CN201810309081.1A
Authority: CN
Inventors: 刘安军; 纪海玉; 于娟
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-04-09
Filing date: 2018-04-09
Publication date: 2020-07-17
Anticipated expiration: 2038-04-09
Also published as: CN108359024A

Abstract

The invention relates to an astragalus alcohol-soluble oligosaccharide, which is α -pyranose, and the preparation method comprises the steps of slicing astragalus roots, adding the sliced astragalus roots into distilled water, soaking at room temperature, carrying out suction filtration, taking the supernatant, centrifuging, removing the precipitate, concentrating by a vacuum rotary evaporator to obtain an astragalus concentrated solution, adding an absolute ethanol solution into the obtained concentrated solution, uniformly mixing, standing overnight in a refrigerator, centrifuging, removing the precipitate, carrying out rotary evaporation to remove ethanol, dialyzing by a dialysis bag, and freeze-drying to obtain the astragalus alcohol-soluble oligosaccharide.

Description

An ethanol soluble oligosaccharide containing radix astragali, and its preparation method and anti-tumor application

Technical Field

The invention belongs to the field of research on the activity of astragalus polysaccharide, relates to research on antitumor activity, and particularly relates to an astragalus alcohol-soluble oligosaccharide, a preparation method thereof and application thereof in antitumor.

Background

Astragalus polysaccharides have various biological functions such as regulating immunity, resisting oxidation, resisting cancer, resisting diabetes, resisting inflammation, resisting virus, protecting liver, resisting atherosclerosis, promoting hematopoiesis and protecting nerve. The astragalus polysaccharide has wide source, low toxic and side effect, no residue in vivo and no drug resistance, so the astragalus polysaccharide has great development prospect in the fields of medicines and nutritional foods.

The method for separating and extracting astragalus polysaccharide is more. At present, the extraction technology of astragalus polysaccharide mainly comprises a water boiling extraction method, an alkaline water extraction method, ultrasonic waves, a microwave-assisted extraction method and the like, wherein a water-soluble alcohol precipitation method is adopted to separate and purify polysaccharide insoluble in ethanol and research various biological activities of the polysaccharide, including anti-tumor, anti-oxidation, anti-bacterial and the like. However, the polysaccharide obtained by the method has low yield, wide molecular weight distribution, higher content of components without biological activity and poorer water solubility. However, low molecular weight polysaccharides dissolved in ethanol have not been considered in the past research and development, and thus waste of resources has been caused.

The oligosaccharide with very uniform molecular weight distribution is found in the ethanol discarded by a water-soluble alcohol precipitation method, has simple composition and good water solubility, and has biological activity of obviously enhancing immunity and resisting tumors in vivo through animal experiments.

Disclosure of Invention

The invention aims to provide an alcohol-soluble astragalus anti-tumor oligosaccharide and a preparation method thereof aiming at the defects of complex structure, low yield, low activity and the like of polysaccharide in the existing water-soluble alcohol precipitation technology.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

an astragalus root alcohol soluble oligosaccharide is α -pyranose.

A method for preparing an astragalus alcohol-soluble oligosaccharide comprises the following steps:

(1) slicing radix astragali, adding into distilled water, soaking at room temperature, vacuum filtering, collecting supernatant, centrifuging, removing precipitate, and concentrating with vacuum rotary evaporator to obtain radix astragali concentrated solution;

(2) and (2) adding an absolute ethanol solution into the concentrated solution obtained in the step (1), uniformly mixing, standing overnight in a refrigerator, centrifuging, removing the precipitate, then performing rotary evaporation to remove ethanol, dialyzing by using a dialysis bag, and freeze-drying to obtain the astragalus alcohol-soluble oligosaccharide.

And (2) soaking for 12-24 hours at room temperature in the step (1).

And (2) repeatedly extracting the filter residue after centrifugation in the step (1) for 2-4 times, mixing the filter residue with the supernatant obtained by the first extraction, and concentrating.

And (3) setting the temperature of the refrigerator to be 3-6 ℃.

And the dialysis bag in the step (2) is a 1000Da dialysis bag.

An application of ethanol-soluble oligosaccharide containing radix astragali in the anti-tumor field is provided.

The invention has the advantages and positive effects that:

1. the invention provides an astragalus alcohol-soluble oligosaccharide which is α -pyranose, has the molecular weight of 2107Da, has the characteristic of typical polysaccharide, contains a small amount of uronic acid and a very small amount of protein, and the monosaccharide comprises arabinose, galactose, glucose and mannose (1.00: 1.60: 4.90: 3.02), and the tumor inhibition rate of a mouse with H22 tumor can reach 52.22% when the intrabody anti-tumor experiment of the astragalus alcohol-soluble oligosaccharide shows that the intrabody anti-tumor dose of the oligosaccharide is 200 mg/kg.

2. The invention efficiently utilizes alcohol-soluble waste in the water-soluble alcohol precipitation process to extract and separate oligosaccharide in the alcohol-soluble waste, and the result shows that the alcohol-soluble oligosaccharide has high yield, simple structure, strong biological activity and high production quality.

Drawings

FIG. 1 is an infrared spectrum of an alcohol soluble oligosaccharide of Astragalus membranaceus of the present invention;

FIG. 2 is a high performance liquid chromatogram of the present invention;

FIG. 3(a) is an ion chromatogram of monosaccharide and uronic acid standards;

in the figure: 1. fucose 2, arabinose 3, galactose 4, glucose 5, mannose 6, xylose 7, fructose 8, galacturonic acid 9, glucuronic acid

FIG. 3(b) is an ion chromatogram of an alcohol-soluble oligosaccharide of Astragalus;

in the figure: 1. arabinose 2, galactose 3, glucose 4, mannose 5, galacturonic acid 6, glucuronic acid

FIG. 4 is a mass spectrometric view of an alcohol-soluble oligosaccharide of Astragalus of the present invention;

FIG. 5 shows the preparation of alcohol soluble oligosaccharides with astragalus root of the present invention¹H spectrum;

FIG. 6 shows the preparation of alcohol soluble oligosaccharides with astragalus root of the present invention¹³C spectrum;

FIG. 7 shows the effect of the alcohol soluble oligosaccharides on the body weight of mice;

FIG. 8(a) is a graph of the effect of the present invention on mouse peripheral blood lymphocyte subsets (CD3/CD19 scatter plot);

FIG. 8(b) is a graph of the effect of the present invention on mouse peripheral blood lymphocyte subsets (CD4/CD8 scatter plot);

FIG. 9 shows the effect of the present invention on mouse thymic lymphocyte subsets;

FIG. 10(a) is H & E staining of mouse thymus (4X) (blank);

FIG. 10(b) is H & E staining of mouse thymus (4X) (model group);

fig. 10(c) is H & E staining of mouse thymus (4X) (low dose group);

fig. 10(d) is H & E staining of mouse thymus (4X) (high dose group);

FIG. 11(a) is H & E staining of mouse spleen (4X) (blank);

FIG. 11(b) is H & E staining of mouse spleen (4X) (model group);

fig. 11(c) is H & E staining of mouse spleen (4 ×) (low dose group);

fig. 11(d) H & E staining of mouse spleen (4X) (high dose group);

FIG. 12 is a graph of the effect of astragalus membranaceus alcohol-soluble oligosaccharides on NK cell activity;

FIG. 13 shows the effect of astragalus membranaceus alcohol-soluble oligosaccharides on phagocytic ability of macrophages in tumor-bearing mice.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments, which are illustrative only and not limiting, and the scope of the present invention is not limited thereby.

A method for preparing ethanol soluble oligosaccharide from radix astragali for resisting tumor comprises slicing radix astragali 100g, soaking in 1000ml distilled water at room temperature for 12 hr, vacuum filtering, collecting supernatant, centrifuging at 3500r/min for 15min, and removing precipitate. Extracting the filter residue after suction filtration once again, mixing the two obtained supernatants, concentrating under a vacuum condition at 50 ℃ by a rotary evaporator to obtain astragalus concentrate, cooling to room temperature, adding 4 times of anhydrous ethanol, mixing uniformly, standing overnight in a refrigerator at 4 ℃, centrifuging at 3500r/min for 15min, removing the precipitate, performing rotary evaporation on the supernatant to remove ethanol, dialyzing for 48h at low temperature by using a 1000Da cellulose dialysis bag to remove inactive substances such as small molecular monosaccharides and the like, and freeze-drying to obtain the astragalus alcohol-soluble oligosaccharide.

Detecting the protein content in the astragalus alcohol-soluble oligosaccharide by adopting a Coomassie brilliant blue method, and calculating to obtain that the protein content in the astragalus alcohol-soluble oligosaccharide is 0.5%. The content of uronic acid in the astragalus alcohol-soluble oligosaccharide is measured by adopting a carbazole sulfuric acid colorimetric method, and the content of uronic acid in the astragalus alcohol-soluble oligosaccharide is calculated to be 10.67%.

The polysaccharide structures were analyzed using a Fourier Infrared spectrometer and the results are shown in FIG. 1. It can be seen that the samples had wavelengths of 3405, 2927 and 1420cm^-1The peaks are respectively a hydroxyl absorption peak, a C-H stretching vibration peak and a C-H bending vibration peak, and are characteristic absorption peaks of the polysaccharide, which indicates that the detection sample has obvious polysaccharide characteristics. 1676cm^-1And 1630cm^-1Is positioned at an O-H bending vibration peak; at a wavelength of 1138cm^-1And 1050cm^-1Absorption peaks are respectively C-O-C and C-O-H stretching vibration peaks in polysaccharide molecules, which indicates that C-O bonds exist in sample molecules, namely that pyranose rings exist in the sample molecules; 923cm^-1The pyranoid ring, which is a sugar, vibrates at 866cm^-1The absorption peak of (2) is the absorption peak of the G-H vertical bond of α -type pyranose ring, the result initially shows that the astragalus alcohol-soluble oligosaccharide is α -type pyranose.

The molecular weight of the astragalus membranaceus alcohol-soluble oligosaccharide was measured by high performance liquid chromatography, and the results are shown in fig. 2. As can be seen from the figure, the absorption peak of the polysaccharide sample has a peak time of 17.806min, and only one peak, which indicates that the astragalus alcohol-soluble oligosaccharide is a purified saccharide with uniform molecular weight, and the molecular weight of the astragalus alcohol-soluble oligosaccharide is about 2000Da according to the standard formula.

The monosaccharide components of the polysaccharides were analyzed using ion chromatography, and the results are shown in fig. 3. Fig. 3(a) is an ion chromatogram of monosaccharide and uronic acid standards, with the order of peaks from left to right: fucose, arabinose, galactose, glucose, mannose, xylose, fructose, galacturonic acid and glucuronic acid. FIG. 3(b) is an ion chromatogram of an astragalus membranaceus alcohol-soluble oligosaccharide, comparing FIG. 3(a), showing that the monosaccharide composition of the astragalus membranaceus alcohol-soluble oligosaccharide is: arabinose, galactose, glucose, mannose, and the molar ratio of each monosaccharide in the sample is: arabinose: galactose: glucose: mannose 1.00: 1.60: 4.90: 3.02.

the molecular weight of the polysaccharide is further determined by adopting an MA L DI-TOF mass spectrometer because the molecular weight of a dextran T series standard adopted by the liquid phase is larger, and the result is shown in figure 4.

By passing¹H nuclear magnetic resonance spectrum and¹³c nmr spectroscopy analyses the H and C atom distributions of the polysaccharide to infer the likely structure of the polysaccharide. The results of NMR analysis are shown in fig. 5 and 6.

FIG. 5 is of polysaccharides¹H spectrum, generally speaking, the signal peak of anomeric proton of sugar is mainly considered to be in the region of 4.3-5.9, the proton signal of α -glucoside anomeric carbon is in the region of 5.0-6.0. two weaker signal peaks at 5.325 and 5.335 in the figure indicate that the α type pyranose exists in the polysaccharide, which is consistent with the infrared result, and D at 4.723₂The solvent peak of O, probably due to the lower concentration of polysaccharide in the polysaccharide solution, results in a higher intensity of the solvent peak; the signal peak area of 3.5-4.1 probably 2H-5H.

FIG. 6 is of polysaccharide¹³And (4) C spectrum. The polysaccharides can be seen from the figure¹³The peak area of the signal of the C spectrum is mainly between 60 and 110, compared with¹H spectrum, its signal range is wider. In general terms, the amount of the solvent to be used,¹³the region of the anomeric carbon signal peak of the spectrum C is mainly between 90 and 110, and the polysaccharide can be seen to have 5 main signal peaks in the range of 90 to 110, and in the range of 98 to 103, the anomeric carbon signal peak region of α -pyranose shows that α pyranose is present in the polysaccharide, and the infrared sum¹The results of the H spectrum are identical; between 675 is the C2-C5 overlap region of polysaccharide, among which, between 60-62 is the signal peak of C6.

The in vivo anti-tumor effect of the astragalus alcohol-soluble oligosaccharide is analyzed as follows:

first, collection of basic physiological indexes of mouse

1. Weight change

Mice were divided into a blank group, a model group, a low dose group and a high dose group, and after two weeks of continuous gavage, right lower limb axillary injection inoculation H was performed on each mouse₂₂The hepatoma carcinoma cells are continuously perfused for 4 weeks, and the liver carcinoma cells are weighed once every 5 days during the test period, and the results are shown in fig. 7, the weight of the mice slowly rises between groups in two weeks before the perfusion, the groups have no obvious difference, and the hair and the eating conditions of the mice are basically similar, which indicates that the astragalus alcohol-soluble oligosaccharides do not cause adverse effects on the body health of the mice; however, after 2 weeks of gastric gavage, the body weight of the model group mice was significantly increased compared to the blank group mice, which may be due to the malignant proliferation of tumor cells, resulting in the increase of tumor volume and thus a large increase in body weight; and the weight growth trend of the mice in the gavage group (low dose group and high dose group) is closer to that of the blank group because the growth of tumors is inhibited by the gavage polysaccharide. This shows that the astragalus alcohol-soluble oligosaccharide can maintain the stability of the physiological index of the mouse to a certain extent and inhibit the growth of tumor.

2. Change in tumor volume

After 2 weeks of gavage, mice were inoculated with H₂₂Tumor cells, after the appearance of accessible solid tumors, the length and width of the tumors were measured with a vernier caliper every 1 day, and the tumor volume was calculated according to the formula, with the results shown in table 1. After 6 days of tumor inoculation, the mice of the model group firstly have the tumors which can be touched, the time of the mice of the gavage group is later than that of the mice of the model group, along with the increase of days, the tumor volume of the model group is greatly increased, the growth speed is faster and faster, some solid tumors of the mice of the gavage group do not have the appearance, after some solid tumors appear, the volume is slowly increased, the growth speed in the later stage is slow, and the phenomena of regression even appear in some solid tumors, which shows that the astragalus alcohol-soluble oligosaccharide can inhibit the tumor growth to a certain extent and delay the time of the tumors.

TABLE 1 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on average tumor volume and tumor inhibition Rate in mice

Note:^acomparison with model set (p)<0.05)

3. Changes in mouse thymus index and spleen index

Immune organs play an important role in resisting tumors in vivo. The spleen is the largest peripheral immune organ of the human body and contains a large number of important immune cells and immune factors. The thymus is an important organ of cellular immunity and is an important site for T cell maturation. Therefore, the change of the two organ forms can reflect the body condition and the immune response condition of the mouse to a certain degree, and the spleen index and the thymus index can reflect the states of the spleen and the thymus to a certain degree.

The blood was taken from the mouse eyeball, the spleen and thymus were taken after cervical dislocation and weighed and recorded, respectively, and the spleen index and thymus index were calculated, and the results are shown in table 2. As shown in the table, the spleen index of the model group mice is obviously increased (p <0.05) compared with that of the blank group, and the thymus index is reduced (p <0.05), which is probably because the tumor growth damages immune organs of the mice, and the spleen enlargement and inflammation, and thymus atrophy of the mice are caused. Although the spleen index of the mice in the gavage group is also increased compared with that in the blank group (p is less than 0.05), the swelling condition is obviously improved compared with that in the model group, which shows that the gavage polysaccharide can improve the swelling and inflammation of the spleen of the mice. The thymus index of the polysaccharide gastric lavage group mice is reduced (p is less than 0.05) compared with that of the blank group mice, but is increased (p is less than 0.05) compared with that of the model group, which shows that the astragalus alcohol-soluble oligosaccharide can protect immune organs of the mice to a certain extent and reduce the damage of tumors to organisms.

TABLE 2 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on mouse organ index

Note:^acomparison with model set (p)<0.05)；^bComparison with blank group (p)<0.05)

Second, analysis of blood index of mouse

1. Routine detection of mouse blood

The whole-cell blood analyzer analyzes the change of the mouse blood cells, thereby judging the influence of the astragalus alcohol-soluble oligosaccharide on the health condition of the mouse, and the result is shown in table 3. It can be seen from the table that there is no statistical difference in the total number of leukocytes, erythrocytes, hemoglobin and platelets in each group of mice, which indicates that the astragalus alcohol-soluble oligosaccharides have no influence on the health status of the mice and do not produce toxic effects on the host animals.

TABLE 3 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on mouse blood routine

2. Analysis of mouse peripheral blood lymphocyte subpopulations

The immunotherapy of cancer mainly stimulates immune cells to further improve the immunity of the organism, thereby achieving the purposes of killing cancer cells and controlling tumor spread. The main immune cells in the human body are lymphocytes, and different lymphocytes can be combined with different monoclonal antibodies. Wherein FITC-CD3⁺Antibody labeling of all T cells, PE-CD19⁺The antibody labels all B cells. T lymphocytes can be further divided into cytotoxic T cells and helper T cells according to their surface molecules, and are respectively associated with PE-CD8⁺And FITC-CD4⁺And (4) combining. Therefore, we used flow cytometry to detect the proportion of lymphocyte subpopulations in peripheral blood of mice, and the analysis results are shown in fig. 8.

In the CD3/CD19 scattergram of FIG. 8(a), the quadrants at the upper left and lower right represent CD3, respectively⁺And CD19⁺The double-stained positive cells of (b) in the CD4/CD8 dot-plot of FIG. 8, the quadrants at the upper left and lower right corners represent CD4, respectively⁺And CD8⁺Double-stained positive cells of (4). The T cell subpopulation distribution is shown in Table 4. The data in Table 4 show the CD3 of the model group and the gavage group⁺Significantly reduced cells compared to the blank group, CD3 in the gavage group⁺The cells were slightly elevated, probably due to the proliferation and metastasis of tumor cells that damaged the immune system of mice, resulting in T-lymphocytes in miceThe secretion of the cells is reduced. In addition, CD4 for model group and intragastric group⁺The cells are obviously lower than those of the blank group, the reduction amplitude of the gavage group is reduced, and even though the high-dose group still has obvious difference compared with the blank group, the high-dose group has obvious increase (p) compared with the model group<0.05); CD8 for model group and intragastric group⁺Cells were significantly reduced compared to the blank group, and CD8 of the gavage group⁺The number of cells slightly increased compared with the number of model groups (p)<0.05); CD19 of model set⁺Cells were significantly elevated (p) relative to the blank<0.05) which may be due to tumor growth, and CD19 in the gavage group⁺The cells were significantly reduced compared to the model group, in which the high dose group CD19⁺The cell number is still different from that of the blank group, but is obviously reduced compared with that of the model group (p)<0.05), which may be due to an impairment of the immune system of the mouse, resulting in an increased secretion of B lymphocytes, enhancing the humoral immunity, but its activity may be decreased, resulting in a weakened immune response in the mouse.

These data indicate that the immune system of mice is greatly damaged due to tumor growth, however, the astragalus alcohol-soluble oligosaccharides can stimulate T lymphocytes and B lymphocytes to some extent and protect immune organs.

TABLE 4 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on mouse peripheral blood lymphocyte subpopulations

Third, mouse thymus index analysis

The thymus is the main endocrine gland of the body and is also an important organ for maintaining the normal immune balance of the body.

1. Mouse thymic lymphocyte subset analysis

The flow cytometry results of detecting the mouse thymic lymphocyte subpopulation ratio are shown in fig. 9.

In FIG. 9, the first quadrant represents CD4⁺And CD8⁺Cells positive for double staining, immature T cellsThe two quadrants indicate that the cells stained positive for CD4 were helper T cells, and the clustering of each cell mass was good and the cell mass of each type was essentially successful. Model group CD4⁺And CD8⁺The proportion of T cells was significantly reduced compared to the blank group, which may be due to the proliferation of tumor cells destroying the structure of thymus, affecting the differentiation of T cells, leading to CD4⁺And CD8⁺A decrease in the proportion of T cells; we can see the CD4 of the gavage group⁺And CD8⁺T cells were significantly reduced compared to the blank group, but increased compared to the model group, even CD4⁺The proportion of T cells is obviously increased, and the differentiation condition of the T cells is improved, which shows that the astragalus alcohol-soluble oligosaccharide can effectively maintain the normal function of thymus gland organs.

TABLE 5 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on mouse thymic lymphocyte subsets

2. Mouse thymus tissue H & E staining

The thymus organ of the human body is a lobate structure, which is enveloped by a thin connective tissue capsule. The capsule is invaginated, dividing the thymus into a number of pseudoleaflets, each of which contains the presence of cortex (exterior) and medulla (interior). Immature or developing T cells are predominantly present in the cortex, and the medulla is the predominant site of mature T cells.

After the mice were sacrificed by decapitation, thymus tissues were taken out and observed. The results showed that the thymus of the mice in the placebo group was larger, while the thymus of the mice in the model group was significantly smaller and almost absent than that of the placebo group, while the thymus of the mice in the gavage group was atrophied as compared with the placebo group, but was atrophied to a lesser extent than that of the model group. Mouse thymus tissues were then paraffin-embedded and the sections were then visualized by H & E staining, the results are shown in fig. 10. It can be seen that the lobules of the thymus were not differentiated significantly in the model group mice, the cortical (dark) proportion was significantly increased compared to the blank group, and the medulla were connected to each other without visible distinct boundaries. Although the cortical area of the thymus of the mice in the low-dose group is still larger than that of the blank group, the medulla still has a slight infection phenomenon, but the boundary limit is obvious compared with that of the model group, and although the ratio of the cortex and the medulla of the high-dose group is still greatly different from that of the blank group, compared with the model group, the thymus is greatly improved, so that the effective protection of the thymus can be seen, and the low-molecular-weight polysaccharide of the astragalus can effectively protect the thymus of the mice.

Fourth, index analysis of mouse spleen

1. Mouse spleen lymphocyte subpopulation analysis

The spleen is the largest peripheral lymphoid organ, and there are a variety of immune cells and immune factors.

The results of the analysis of the mouse spleen lymphocyte subpopulations are shown in Table 6. The spleen lymphocytes of the model mice are all significantly different from those of the blank mice, wherein the CD3 is⁺、CD4⁺、CD8⁺The cell proportion is remarkably reduced, and the CD19⁺The proportion of cells rose significantly. Although CD3 in the Low dose group⁺The cells are obviously increased compared with the model group, but are also obviously different compared with the blank group of mice, and the CD3 is obtained in the high-dose group⁺The cell proportion was closer to that of the blank group; high dose group CD19⁺Although the cell proportion is slightly reduced compared with the model group, the cell proportion has no statistical difference; intragastric group CD4⁺And CD8⁺The proportion of cells was slightly increased compared to the model group, but was still slightly different from the blank group.

TABLE 6 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on spleen lymphocyte subsets in tumor-bearing mice

2. H & E staining of mouse spleen tissue

After the mice were sacrificed by cervical dislocation, spleen tissues were removed and observed. The results show that the spleen of the model group mice is obviously enlarged, the spleen of the blank group mice is smaller, and the spleen of the gastric lavage group mice is enlarged compared with the blank group, but is smaller than the spleen of the model group mice, and even the spleen of the high-dose group mice is closer to the spleen of the blank group. Thereafter, the spleen tissue of the mouse was embedded in paraffin, and then the section was observed by H & E staining, and the results are shown in FIG. 11. The blank group of spleen white (dark) and red (light) marrow regions are clearly visible in the figure and the boundaries of the respective regions are well defined, showing clear marginal zones, whereas the spleen tissue of the model group is more loosely textured, with a significant increase in the area of the red marrow region accompanied by a decrease in the area of the white marrow region and no clear marginal zones. The red marrow areas of the mice in the gavage group are enlarged, and the marginal areas are visible; in addition, spleen of the high-dose mice can show more obvious white marrow and red marrow boundaries, and the proportion of the red marrow and the white marrow is closer to that of the blank group. Therefore, the spleen of the model group mouse is seriously damaged due to the growth of the tumor, the astragalus alcohol-soluble oligosaccharide can effectively protect the spleen tissue and improve the spleen lesion, and the proportion of red marrow and white marrow is better displayed.

3. Analysis of mouse spleen lymphocyte transformation Capacity

The experimental results are shown in table 7, and the lymphocyte stimulation index of the model group mouse is obviously reduced compared with that of the blank group, which is probably because malignant proliferation of tumor cells greatly damages the immune system of the mouse, so that the tumor cells cannot be effectively killed, the immunity of the body is obviously reduced, and the lymphocyte stimulation index of the mouse is reduced. However, the stimulation index of the mice in the gavage group is obviously increased compared with the mice in the model group and is closer to that in the blank group (p is less than 0.05), which shows that the astragalus alcohol-soluble oligosaccharide can promote the proliferation of T cells and B cells and stimulate the immune system of the mouse body.

TABLE 7 Effect of Astragalus membranaceus alcohol-soluble oligosaccharides on the proliferative capacity of lymphocytes from tumor-bearing mice

4 mouse spleen NK cell Activity assay

NK cells are a congenital immune cell of the body, participate in the first line of defense of the body against cancer cells, and can directly attack tumor cells. Therefore, the results of the assay using the MTT method for NK cell activity are shown in FIG. 12. The results indicate that the model group had significantly reduced NK cell activity compared to the blank group, probably due to inhibition of NK cell activity by tumor cell proliferation. And the NK cell activity of the mice in the gavage group is obviously reduced compared with that of the blank group, but is obviously improved compared with that of the model group, which shows that the astragalus alcohol-soluble oligosaccharide can protect the spleen of the mice and increase the NK cell activity of organisms. The astragalus polysaccharide can quantitatively promote the activity of NK cells.

Fifth, analysis of phagocytic neutral Red ability of mouse macrophage

Macrophages are important cells of the innate immunity of the body and are important immune cells in the biological defense system against tumor growth. The activated macrophage can directly participate in antigen presentation to kill tumor cells. Therefore, the activity of macrophage can reflect the immune function of the body to a certain extent. The neutral red colorimetric method is a commonly used detection method for rapidly evaluating the phagocytic capacity of macrophages. Macrophages in abdominal cavities of the mice of each group are respectively extracted and cultured together with neutral erythrocytes, and the phagocytic capacity is determined by an MTT method, and the experimental result is shown in figure 13. The macrophage activity of the model group is obviously reduced compared with that of the blank group, while the macrophage activity of the lavage group is also obviously reduced compared with that of the blank group, but is obviously increased compared with the model group (p < 0.05). This shows that the astragalus alcohol-soluble oligosaccharide can obviously increase the activity of macrophage and stimulate the immune system of the organism to resist tumor cells.

Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Claims

1. A method for preparing alcohol soluble astragalus oligosaccharide is characterized in that the oligosaccharide is α -pyranose, and the preparation method comprises the following steps:

2. The method of preparing an astragalus membranaceus alcohol-soluble oligosaccharide according to claim 1, wherein the method comprises the following steps: and (2) soaking at room temperature for 12-24 h.

3. The method of preparing an astragalus membranaceus alcohol-soluble oligosaccharide according to claim 1, wherein the method comprises the following steps: and (2) repeatedly extracting the filter residue after centrifugation in the step (1) for 2-4 times, mixing the filter residue with the supernatant obtained by the first extraction, and concentrating.

4. The method of preparing an astragalus membranaceus alcohol-soluble oligosaccharide according to claim 1, wherein the method comprises the following steps: and (2) setting the temperature of the refrigerator to be 3-6 ℃.

5. The method of preparing an astragalus membranaceus alcohol-soluble oligosaccharide according to claim 1, wherein the method comprises the following steps: the dialysis bag in the step (2) is a 1000Da dialysis bag.