CN110218265B - Middle molecular weight section astragalus polysaccharide, preparation method and application thereof - Google Patents
Middle molecular weight section astragalus polysaccharide, preparation method and application thereof Download PDFInfo
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- CN110218265B CN110218265B CN201910561282.5A CN201910561282A CN110218265B CN 110218265 B CN110218265 B CN 110218265B CN 201910561282 A CN201910561282 A CN 201910561282A CN 110218265 B CN110218265 B CN 110218265B
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- molecular weight
- astragalus
- polysaccharide
- astragalus polysaccharide
- acid
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Images
Classifications
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a medium molecular weight section astragalus polysaccharide, a preparation method and application thereof, belonging to the technical field of biological medicine manufacturing. The astragalus polysaccharide with middle molecular weight segment is obtained by hydrolyzing astragalus crude polysaccharide, and the molecular weight range of the astragalus polysaccharide with middle molecular weight segment is 103‑104Dalton, the invention also discloses a preparation method of the middle molecular weight section astragalus polysaccharide, which is to perform acid hydrolysis on the astragalus crude polysaccharide to obtain the middle molecular weight section astragalus polysaccharide. The invention also discloses application of the medium molecular weight astragalus polysaccharide in preparing medicines or foods for improving intestinal mucosa immunity and intestinal microecosystem functions. The astragalus polysaccharide with medium molecular weight segment produced after the hydrolysis of the astragalus polysaccharide has better effect than the natural astragalus polysaccharide in the aspect of regulating the intestinal mucosa immunity and intestinal microecosystem, and enhances the health care effect of the astragalus polysaccharide. The acid hydrolysis process of the astragalus polysaccharide is simple and is easy to realize industrial production.
Description
Technical Field
The invention relates to the technical field of biological medicine manufacturing, in particular to a preparation method and application of astragalus polysaccharide with medium molecular weight segment.
Background
The astragalus is one of the most widely used traditional Chinese medicinal materials in clinical application of traditional Chinese medicine, and is advocated by doctors of all ages due to the definite curative effect and wide application range of the astragalus. The astragalus root is sweet and warm in nature and taste, and has the effects of tonifying qi and invigorating yang, benefiting wei and protecting superficies, inducing diuresis to alleviate edema, supporting sores and promoting granulation and the like; has better curative effect on diseases of circulatory system, nervous system, digestive system, respiratory system, endocrine and blood system. Pharmacological research shows that the astragalus extract or monomer compound has definite functions in the aspects of anti-inflammation, immunoregulation, anti-tumor, myocardial ischemia protection, diabetes, oxidation resistance, aging resistance and the like. From the perspective of modern research, the main function of astragalus root is to regulate immune function, and the regulation of astragalus root on immune function is closely related to the action of qi-tonifying and exterior-securing in its traditional application, which has been said in huangdi's internal channel & lingshu: the "defensive qi failing to nourish the body and pathogenic qi residing in it" clearly indicates that the defensive qi is a defense mechanism against pathogenic qi in the body, and modern researches suggest that the weak defensive qi is mainly manifested as low immune function, so the core efficacy of astragalus root is the influence on the immune function. A great deal of research in the prior art proves that astragalus polysaccharide is one of the main active components of astragalus. The molecular weight of the natural astragalus polysaccharide is high, the average molecular weight is more than 10 ten thousand, and the natural astragalus polysaccharide cannot be normally digested and absorbed by a human body, so that the application provides the astragalus polysaccharide with a medium molecular weight segment, a preparation method and application thereof.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a medium molecular weight fraction astragalus polysaccharide, a preparation method and application thereof.
The invention aims to provide a medium molecular weight astragalus polysaccharide obtained by hydrolyzing crude astragalus polysaccharide, wherein the molecular weight range of the medium molecular weight astragalus polysaccharide is 103-104And D, dalton.
The second object of the present invention is to provide a method for preparing medium molecular weight fraction astragalus polysaccharides, which comprises the following steps: dissolving radix astragali crude polysaccharide in hot water, adding acid, heating in water bath, adding alkali solution to adjust pH to neutral, concentrating and drying the hydrolysate, pulverizing, and sieving to obtain radix astragali polysaccharide hydrolysate with the content of radix astragali polysaccharide in medium molecular weight segment not less than 50%.
Preferably, the hydrolysate is further dialyzed after being concentrated and before being dried, so that the purified astragalus polysaccharide with the medium molecular weight segment is obtained.
Preferably, the acid is an organic acid or an inorganic acid capable of hydrolyzing a glycosidic bond.
Preferably, the acid is hydrochloric acid, the concentration of the hydrochloric acid in the astragalus crude polysaccharide solution is adjusted to be 0.03-0.30 mol/L, the water bath heating temperature is 10-100 ℃, and the time is 1-8 hours.
Preferably, the acid is trifluoroacetic acid, the concentration of the trifluoroacetic acid in the crude astragalus polysaccharide solution is adjusted to be 0.05-0.30 mol/L, the water bath heating temperature is 10-100 ℃, and the time is 1-8 hours.
Preferably, the method for extracting the astragalus crude polysaccharide comprises the following steps: pulverizing radix astragali, extracting with warm water, concentrating the extractive solution, removing protein, centrifuging, concentrating the supernatant, washing with 80% ethanol, and precipitating to obtain radix astragali crude polysaccharide.
The invention also aims to provide the application of the astragalus polysaccharide with the medium molecular weight segment in the preparation of medicines or foods for improving the intestinal mucosa immunity and the intestinal microecosystem function.
Compared with the prior art, the invention has the beneficial effects that: the molecular weight range of the astragalus polysaccharide with the medium molecular weight section is 103～104The molecular diffusion speed is high between dalton, the molecular diffusion speed is easy to be utilized by intestinal bacteria, the molecular diffusion speed is easy to interact with intestinal mucosa cells, and the functions of regulating intestinal mucosa immunity and an intestinal microecosystem can be better realized;
the invention hydrolyzes natural astragalus polysaccharide to obtain astragalus polysaccharide with middle molecular weight segment, and the method of acid hydrolysis can hydrolyze the astragalus polysaccharide into astragalus polysaccharide with middle molecular weight segment, and can not degrade the astragalus polysaccharide into monosaccharide or oligosaccharide completely, the content of the astragalus polysaccharide with middle molecular weight segment in the astragalus polysaccharide hydrolysate obtained by the method of acid hydrolysis is not less than 50%;
experiments prove that the astragalus polysaccharide with medium molecular weight segment generated after the astragalus polysaccharide is hydrolyzed has better effect than natural astragalus polysaccharide in the aspect of regulating intestinal mucosa immunity and intestinal microecosystem, and enhances the health care effect of the astragalus polysaccharide. The acid hydrolysis process of the astragalus polysaccharide is simple and is easy to realize industrial production.
Drawings
FIG. 1 shows the effect of APS and DAPS treatments of the present invention on the immunosuppressive mouse small intestinal tissue ZO-1
n＝3)；
FIG. 2 is the micro-ecosystem diversity index and abundance index (n is 3) of the mouse intestinal flora of the present invention;
FIG. 2a is the Chao index of intestinal flora of mice treated with APS and DAPS according to the present invention;
FIG. 2b is the Ace index of the intestinal flora of mice treated with APS and DAPS according to the present invention;
FIG. 2c is a Shannon index of intestinal flora of mice treated with APS and DAPS according to the present invention;
FIG. 2d is a Simpson index of intestinal flora of mice treated with APS and DAPS according to the present invention;
FIG. 3 is a PCoA analysis graph of intestinal flora of mice treated with APS and DAPS according to the present invention (n-3);
FIG. 4 shows the effect of APS and DAPS treatment on SCFA, a metabolite of mouse intestinal flora
n＝5)。
FIG. 4a is a graph showing the effect of APS and DAPS treatments on the metabolite acetate of the mouse intestinal flora.
FIG. 4b shows the effect of APS and DAPS treatment on propionic acid, a metabolite of the mouse intestinal flora.
FIG. 4c is a graph showing the effect of APS and DAPS treatments on butyric acid, a metabolite of the mouse intestinal flora.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings and examples, but it should be understood that the scope of the present invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The raw materials and auxiliary materials used for preparing 1000 tablets containing the active ingredient and the astragalus polysaccharide with medium molecular weight segment as an example and the mass ratio are as follows:
1. extracting astragalus polysaccharide: pulverizing radix astragali, extracting with water, concentrating the extractive solution, removing protein by Sevag method, centrifuging, concentrating the supernatant, precipitating with 80% ethanol, and washing the precipitate to obtain radix astragali crude polysaccharide;
2. acid hydrolysis of crude astragalus polysaccharides: dissolving radix astragali crude polysaccharide in hot water, adding hydrochloric acid to concentration of 0.16mol/L (mass percentage concentration of 0.05% w/w), water bathing at 80 deg.C for 3 hr, adding equivalent NaOH solution to adjust pH to neutrality, concentrating the hydrolysate to extract, drying, pulverizing, and sieving to obtain extract rich in 10 molecular weight3～104Middle molecular weight fraction of Astragalus polysaccharides in Dalton.
Taking 1000 tablets containing the astragalus polysaccharide with the medium molecular weight as an active ingredient as an example, the raw materials and auxiliary materials are as follows by mass ratio: 150g of astragalus polysaccharide product rich in medium molecular weight segment, 1450g of corn starch and 5g of magnesium stearate are carried out according to the conventional process of pharmaceutical tablets, each tablet weighs 0.3g, and each tablet contains 150mg of astragalus polysaccharide in medium molecular weight segment. The usage and dosage are as follows: orally administered in 3 tablets each time, three times a day, and reduced for children.
Example 2
Taking 1000 bags of astragalus polysaccharide granules containing active ingredients as medium molecular weight segments as an example for preparation, the raw materials and auxiliary materials and the mass ratio are as follows:
1. extracting astragalus polysaccharide: pulverizing radix astragali, extracting with water, concentrating the extractive solution, removing protein by Sevag method, centrifuging, concentrating the supernatant, precipitating with 80% ethanol, and washing the precipitate to obtain radix astragali crude polysaccharide;
2. acid hydrolysis of crude astragalus polysaccharides: dissolving the crude radix astragali polysaccharide in hot water, adding trifluoroacetic acid to adjust the concentration to 0.1mol/L (0.8% w/w), and water-bathing at 80 deg.C for 3 h; water bath at 80 deg.C for 3 hr, adding equivalent NaOH solution to adjust pH to neutral, concentrating the hydrolysate, dialyzing with dialysis bags with molecular weight cut-off of 1KD and 10KD, drying, pulverizing, and sieving to obtain purified product with molecular weight of 103～104Middle molecular weight fraction of Astragalus polysaccharides in Dalton.
Taking 1000 bags of granules containing active ingredients as astragalus polysaccharide with medium molecular weight as an example, the granules comprise the following raw materials and auxiliary materials in percentage by mass: 450g of astragalus polysaccharide product with medium molecular weight, 2000g of sucrose powder and 650g of dextrin. The preparation is carried out according to the conventional process of the pharmaceutical granules, each bag weighs 3g, and contains 450mg of astragalus polysaccharide with medium molecular weight. The usage and dosage are as follows: orally taken, one bag each time and three times a day.
Example 3
The raw materials and auxiliary materials used for preparing 1000 capsules containing the astragalus polysaccharide with the active ingredient as the medium molecular weight segment and the mass ratio are as follows:
1. extracting astragalus polysaccharide: pulverizing radix astragali, extracting with water, concentrating the extractive solution, removing protein by Sevag method, centrifuging, concentrating the supernatant, precipitating with 80% ethanol, and washing the precipitate to obtain radix astragali crude polysaccharide;
2. acid hydrolysis of crude astragalus polysaccharides: dissolving radix astragali crude polysaccharide in hot water, adding hydrochloric acid to adjust concentration to 0.16mol/L (0.5%), water-bathing at 80 deg.C for 6 hr, adding equivalent NaOH solution to adjust pH to neutral, concentrating the hydrolysate to extract, drying, pulverizing, and sieving to obtain extract rich in polysaccharide with molecular weight of 103～104Middle molecular weight fraction of Astragalus polysaccharides in Dalton.
Taking 1000 bags of granules containing active ingredients as astragalus polysaccharide with medium molecular weight as an example, the granules comprise the following raw materials and auxiliary materials in percentage by mass: 150g of astragalus polysaccharide product with medium molecular weight segment and 150g of corn starch. The preparation is carried out according to the conventional process of pharmaceutical capsules, each capsule weighs 3g and contains 150mg of astragalus polysaccharide with medium molecular weight. The usage and dosage are as follows: orally administered 2 capsules each time, three times a day.
Example 4
The raw materials and auxiliary materials used for preparing 1000 granules of the astragalus polysaccharide oral liquid containing active ingredients as medium molecular weight segments and the mass ratio are as follows:
1. extracting astragalus polysaccharide: pulverizing radix astragali, extracting with water, concentrating the extractive solution, removing protein by Sevag method, centrifuging, concentrating the supernatant, precipitating with 80% ethanol, and washing the precipitate to obtain radix astragali crude polysaccharide; 2. Acid hydrolysis of crude astragalus polysaccharides: dissolving radix astragali crude polysaccharide in hot water, adding trifluoroacetic acid to adjust concentration to 0.10mol/L (0.5%), water-bathing at 60 deg.C for 6 hr, adding equivalent NaOH solution to adjust pH to neutrality, concentrating the hydrolysate, dialyzing with dialysis bags with molecular weight cut-off of 1KD and 10KD, drying, pulverizing, and sieving to obtain purified polysaccharide with molecular weight of 103～104Middle molecular weight fraction of Astragalus polysaccharides in Dalton.
Taking 1000 prepared oral liquid containing astragalus polysaccharide with middle molecular weight as active ingredient as example, the raw materials and auxiliary materials and the mass ratio are as follows: 450g of astragalus polysaccharide with medium molecular weight, 6000g of sucrose powder, 10g of benzoic acid and 10000ml of distilled water. The preparation is carried out according to the conventional process of the pharmaceutical oral liquid, each 10ml contains 450mg of astragalus polysaccharide with medium molecular weight segment. The usage and dosage are as follows: orally administered 1 pill three times a day.
Example 5
Taking the preparation of bread containing astragalus polysaccharide with medium molecular weight as an active ingredient as an example, the process is as follows:
1. extracting astragalus polysaccharide: pulverizing radix astragali, extracting with water, concentrating the extractive solution, removing protein by Sevag method, centrifuging, concentrating the supernatant, precipitating with 80% ethanol, and washing the precipitate to obtain radix astragali crude polysaccharide;
2. acid hydrolysis of crude astragalus polysaccharides: dissolving radix astragali crude polysaccharide in hot water, adding hydrochloric acid to concentration of 0.16mol/L (mass percentage concentration of 0.05% w/w), water bathing at 80 deg.C for 3 hr, adding equivalent NaOH solution to adjust pH to neutrality, concentrating hydrolysate,dialyzing with dialysis bags with molecular weight cut-off of 1KD and 10KD, drying, pulverizing, sieving to obtain purified product with molecular weight of 103～104Middle molecular weight fraction of Astragalus polysaccharides in Dalton.
The bread containing the astragalus polysaccharide with the medium molecular weight as the active ingredient is prepared by taking the following raw materials and auxiliary materials in parts by weight: 5.4g of astragalus polysaccharide product with medium molecular weight, 930g of flour, 50g of vegetable oil and 20g of active yeast. Making bread according to the conventional bread making process. The bread contains 250g of astragalus polysaccharide with medium molecular weight segment and 1.35g of astragalus polysaccharide per person per day.
The astragalus polysaccharide with medium molecular weight can also be added into food such as biscuits, cakes and the like, and the consumption of the astragalus polysaccharide with medium molecular weight is 1-2 g per person per day.
We verify the hydrolysis effect of astragalus polysaccharides under different hydrolysis conditions and the stability problem of the hydrolysis process; in order to verify the benefit of the astragalus polysaccharide with the medium molecular weight segment in the aspects of regulating the intestinal mucosa immune function and the intestinal tract microecosystem, the applicant also performs comparison and efficacy test of various process parameters, and the related experiments are as follows:
firstly, acid hydrolysis process research of astragalus polysaccharide:
1.1 preparation of Astragalus polysaccharides
Taking 100g of astragalus dry powder, adding water according to the material-liquid ratio of 1: 10 for reflux extraction, filtering, collecting filtrate, repeating for 3 times, combining the filtrates, centrifuging to obtain supernatant, concentrating the supernatant under reduced pressure to 400ml, then adding ethanol to make the alcohol concentration reach 80%, standing for 24h, filtering to obtain precipitate, drying to obtain crude astragalus polysaccharide, deproteinizing the crude astragalus polysaccharide by a Sevage method, and drying to obtain the astragalus polysaccharide. The protein content in the prepared astragalus polysaccharide is less than 1% by detection of Coomassie brilliant blue method, and the sugar content in the prepared astragalus polysaccharide is 57.40% by detection of phenol-sulfuric acid method (0.5740 +/-0.0174, n is 5).
1.2 detection of polysaccharide molecular weight-high performance gel permeation chromatography:
1.2.1 chromatographic conditions: reprosil 125SEC column, 5 μm, 300X 7.8mm, column temperature 30 deg.C, mobile phase deionized water, flow rate 0.5mL/min, detector ELSD3300 evaporative light scattering detector (Alltech), temperature 100 deg.C. Dextran molecular weight standards (Mw4320, Mw12600, Mw70800, Mw126000, beijing solibao technologies ltd).
It should be noted that, the effective separation range of the Reprosil 125SEC chromatographic column is 3000-500000, so the calculated molecular weight and the actual molecular weight will vary, and considering the research purpose of the experiment, the separation range of the separation column is enough to determine the hydrolysis condition of the astragalus polysaccharide and the yield problem of the astragalus polysaccharide in the medium molecular weight fraction.
1.2.2 preparation of molecular weight Standard Curve
Under the above chromatographic conditions, dextran molecular weight standards (Mw4320, 12600, 70800, 126000, Beijing Soilebao Tech. Co., Ltd.) and glucose samples were used for detection. The retention time (min) is plotted on the abscissa and the logarithm of the relative molecular weight (lg Mw) is plotted on the ordinate.
1.2.3 sample molecular weight detection
Under the chromatographic conditions, the sample is dissolved in deionized water for sampling detection. The sample retention time was substituted into the equation for the standard curve and the sample relative molecular mass (Mw) was calculated.
1.3 study of acid hydrolysis Process of Astragalus polysaccharides
1.3.1 hydrolysis process of astragalus polysaccharide by hydrochloric acid
10mg of astragalus polysaccharide was weighed into a test tube with a stopper, and the influence of time and acid concentration was examined respectively.
According to the pre-experimental results, the hydrochloric acid concentrations are respectively set as: 0.08, 0.160 and 0.320 mol/L; hydrolyzing at 80 deg.C for 5 hr, adjusting pH of the solution to neutral with NaOH solution, freeze drying, dissolving with deionized water, and analyzing molecular weight information by HPLC-ELSD.
1.3.2 hydrolysis process of astragalus polysaccharide trifluoroacetic acid
10mg of astragalus polysaccharide was weighed into a test tube with a stopper, and the influence of time and acid concentration was examined respectively.
According to the results of the preliminary experiments, the trifluoroacetic acid concentrations were set as: 0.05, 0.10 and 0.20 mol/L; hydrolyzing at 80 deg.C for 5 hr, adjusting pH of the solution to neutral with NaOH solution, freeze drying, dissolving with deionized water, and analyzing molecular weight information by HPLC-ELSD.
1.4 acid hydrolysis of Astragalus polysaccharides research results
1.4.1 polysaccharide molecular weight Standard Curve
Dextran standards of different relative molecular masses and glucose were used, according to the 1.2 method. The retention time of the gel on the chromatographic column is measured by an HPSEC-ELSD method, the retention time (t) of each standard is taken as an X axis, lgMw is taken as a y axis, and a regression equation for drawing a standard curve is as follows: y-0.281 x +8.279, R2-0.9839.
TABLE 1 Retention time of different standard dextran molecules
1.4.2 results of hydrochloric acid hydrolysis of Astragalus polysaccharides
The molecular weight composition of the polysaccharides in the hydrolysate was determined after hydrolysis at different hydrochloric acid concentrations and the results were as follows:
TABLE 2 hydrolysis Effect of Astragalus polysaccharides with different hydrochloric acid concentrations
According to the experimental results, the hydrolyzed astragalus polysaccharide mainly comprises three parts of materials with different molecular weight sections, namely high, middle and low, wherein the high molecular weight section is mainly unhydrolyzed astragalus polysaccharide, and the average relative molecular weight is 1.5 multiplied by 105About dalton, the middle molecular weight segment mainly comprises partially hydrolyzed Astragalus polysaccharides, and the molecular weight is 103～104Dalton, and the low molecular weight segment is mainly composed of monosaccharide or oligosaccharide with complete hydrolysis, and the molecular weight is less than 103And D, dalton. The results show that the proportion of the high molecular weight section is gradually reduced and the proportion of the medium and low molecular weight sections is gradually increased along with the gradual increase of the concentration of the hydrochloric acid, when the concentration of the hydrochloric acid is 0.16mol/L, the content of the astragalus polysaccharide in the high molecular weight section is reduced to about 10 percent, and the relative proportion of the polysaccharide in the medium molecular weight section reaches more than 70 percent, which indicates that most of the astragalus polysaccharide is hydrolyzed, and the astragalus polysaccharide with medium and small molecular weight is mainly in the products. When the concentration of the hydrochloric acid is 0.32mol/L, the relative proportion of substances in the low molecular weight section reaches more than 90 percent, and the molecular weight is between 400 and 500 daltons, which indicates that the astragalus polysaccharide is hydrolyzed completely.
Therefore, when the concentration of hydrochloric acid is 0.16mol/L, the hydrolysis temperature is 80 ℃, and the hydrolysis time is 5 hours, the hydrolysis condition is more suitable for preparing the astragalus polysaccharide with the medium molecular weight segment.
1.4.3 results of the Trifluoropolysaccharide trifluoroacetic acid hydrolysis process
The molecular weight composition of the polysaccharides in the hydrolysate was determined after hydrolysis at different trifluoroacetic acid concentrations and the results are as follows:
TABLE 3 hydrolysis Effect of Astragalus polysaccharides at different trifluoroacetic acid concentrations
Similar to the experimental results of hydrochloric acid hydrolysis, the hydrolyzed Astragalus polysaccharides mainly comprise three parts of high, medium and low molecular weight fractions, wherein the high molecular weight fraction is mainly unhydrolyzed Astragalus polysaccharides, and the average relative molecular weight is 1.5 × 105About dalton, the middle molecular weight segment mainly comprises partially hydrolyzed Astragalus polysaccharides, and the molecular weight is 103～104Daltons, while the low molecular weight segment is mainly composed of monosaccharides or oligosaccharides that are hydrolyzed relatively thoroughly, and has a molecular weight below 1000 daltons. As can be seen from the results, with the gradually increasing concentration of trifluoroacetic acid, the proportion of the high molecular weight segment gradually decreases, and the proportion of the medium and low molecular weight segment gradually increases, according to the experimental results, the trifluoroacetic acid concentration is 0.10mol ^ based onAnd in the L period, the content of the astragalus polysaccharide in the high molecular weight section is reduced to about 25 percent, and the relative proportion of the medium molecular weight section reaches more than 60 percent, which shows that most of the astragalus polysaccharide is hydrolyzed, and the relative molecular weights are about 4000 daltons respectively. At a concentration of 0.20mol/L trifluoroacetic acid, the relative proportion of low molecular weight fraction in the product having a molecular weight below 1000 is high, indicating that the hydrolysis is relatively complete.
Therefore, when the concentration of trifluoroacetic acid is 0.10mol/L, the hydrolysis temperature is 80 ℃, and the hydrolysis time is 5h, the hydrolysis condition is more suitable for preparing the astragalus polysaccharide with the medium molecular weight segment.
1.4.4 conclusion:
the concentration of the hydrochloric acid hydrolysis of the astragalus polysaccharide is 0.16 mol/L; the trifluoroacetic acid is preferably 0.10mol/L, and the temperature can be 80 ℃ and the hydrolysis time can be 5h when different acids are hydrolyzed.
1.4.6 discussion
When the astragalus polysaccharide is hydrolyzed completely, the products are mainly oligosaccharides and monosaccharides, the molecular weight is generally lower than 1000 daltons, and the oligosaccharides and monosaccharides have small influence on the mucosal immune function; the hydrolysate has a molecular weight of 103～104Polysaccharides between daltons may have both good mucosal immune regulation and intestinal microecosystem regulation, so the main evaluation index of astragalus polysaccharide hydrolysis process is to increase the molecular weight of hydrolysate to 103～104Relative content of polysaccharides in daltons. In the above experiment, the molecular weight is preferably 103～104The relative content of the astragalus polysaccharide with middle molecular weight of dalton in the total hydrolysate is not less than 50%.
The most main factors influencing the hydrolysis of astragalus polysaccharide are acid concentration, which is too high to cause over-hydrolysis and even total hydrolysis into monosaccharide, and too low acid concentration and too low hydrolysis degree which is 103～104The product of polysaccharides in daltons is too little. The hydrolysis time, the hydrolysis temperature and other conditions have relatively small influence on the hydrolysis degree, and can be adjusted according to the actual production requirements. In addition, the first and second substrates are,the hydrolysis of astragalus polysaccharide can select various acids, such as sulfuric acid, phosphoric acid, nitric acid and the like, but theoretically analysis shows that the sulfuric acid and the nitric acid have oxidizing property, the types of hydrolysis products are more, the structure of the products can also be changed, and simultaneously, other particles such as sulfate radicals and nitrate radicals are doped in the products, so that the subsequent functions of the products are influenced, and the environmental pollution is higher. The hydrochloric acid hydrolysis process introduces chloride ions, the boiling point of trifluoroacetic acid is 72.4 ℃, the chlorine ions can be volatilized and removed in the drying process, and the residue in the product has little influence on the functions of future products, so that the selection of hydrochloric acid and trifluoroacetic acid has certain advantages in industrial production.
Stability study of acid hydrolysis process of astragalus polysaccharide
Compared with the enzymatic hydrolysis method, the polysaccharide hydrolyzed by the acid hydrolysis method has high randomness of hydrolysis sites and relatively complex product components, so that whether the product composition is stable under the acid hydrolysis process condition is an important factor related to the product efficacy, and the composition stability condition of the acid hydrolysis product under the optimized process condition is explored. For the present invention, the core requirement of the acid hydrolysis product is that after the astragalus polysaccharide is hydrolyzed, the product is the astragalus polysaccharide with small molecular weight and small polymerization degree, and the astragalus polysaccharide is not excessively hydrolyzed into monosaccharide or oligosaccharide to reduce the immune regulation function of the astragalus polysaccharide. The stability of the reaction hydrolysis process is formed by detecting the molecular weight of the astragalus polysaccharide acid hydrolysis product.
2.1 preparation of astragalus polysaccharide: same as 1.1
2.2 detection of polysaccharide molecular weight-high performance gel permeation chromatography: same as 1.2
2.3 study of stability of Astragalus polysaccharides in acid hydrolysis
2.3.1 study of stability of Astragalus polysaccharides in hydrochloric acid hydrolysis
Weighing 10mg of astragalus polysaccharide, putting the astragalus polysaccharide into a test tube with a plug, and adding hydrochloric acid with the concentration of 0.160 mol/L; hydrolyzing at 80 deg.C for 5 hr, adjusting pH of the solution to neutral with NaOH solution, freeze drying, dissolving with deionized water, and analyzing molecular weight information by HPLC-ELSD. The experiment was repeated three times and the Relative Standard Deviation (RSD) variation in the relative proportions of the molecular weight distribution of the hydrolysate between the different batches was calculated.
2.3.2 study of stability of Astragalus polysaccharides trifluoroacetic acid hydrolysis Process
Weighing 10mg of astragalus polysaccharide, putting the astragalus polysaccharide into a test tube with a plug, and adding trifluoroacetic acid with the concentration of 0.10 mol/L; hydrolyzing at 80 deg.C for 5 hr, adjusting pH of the solution to neutral with NaOH solution, freeze drying, dissolving with deionized water, and analyzing molecular weight information by HPLC-ELSD. The experiment was repeated three times and the Relative Standard Deviation (RSD) variation of the molecular weight distribution of the hydrolysate between different batches was calculated.
2.4 results
2.4.1 the retention time on the column was determined by the HPSEC-ELSD method as in 1.4.1, with retention time (t) for each standard on the X-axis and lgMw on the y-axis, and the regression equation for plotting the standard curve was: y ═ 0.281x +8.279, R2＝0.9839。
2.4.2 study of stability of Astragalus polysaccharides in hydrochloric acid hydrolysis
As shown in Table 5, from the changes of the molecular weight of the astragalus polysaccharide hydrolysate and the relative content of each molecular weight segment, the RSD value is smaller, wherein the peak 2 is a desired product, the variation of the molecular weight range is relatively larger, but the relative content of the peak 2 is still within the desired range, so that the process has better stability and can meet the preparation requirement of the astragalus polysaccharide hydrolysate.
TABLE 5 study of stability of Astragalus polysaccharides by hydrochloric acid hydrolysis
2.4.3 study of stability of Astragalus polysaccharides trifluoroacetic acid hydrolysis Process
As shown in Table 6, similar to the results of the hydrochloric acid hydrolysis process of Astragalus membranaceus, the RSD value of the relative content and molecular weight change of peak 2 is small, the average molecular weight is about 3000 daltons, and the relative content accounts for more than 60% of the total product, which indicates that the process has good stability and can meet the preparation requirement of the astragalus polysaccharide hydrolysate.
TABLE 6 study of stability of Astragalus polysaccharides trifluoroacetic acid hydrolysis Process
2.4.3 conclusion
The above repeatability experiments prove that the hydrochloric acid hydrolysis process and the trifluoroacetic acid hydrolysis process of the astragalus polysaccharide have good repeatability, and are suitable for industrial production and preparation of the astragalus polysaccharide with medium molecular weight.
Experimental study on influence of acid hydrolysis product of astragalus polysaccharide on immunosuppressed mouse mucosal immune function
3.1 Experimental materials: the preparation method of Astragalus polysaccharides is the same as 1.1. The preparation method of Astragalus polysaccharides acid hydrolysate is 2.3.1, and the Astragalus polysaccharides acid hydrolysate is dialyzed with dialysis bags with molecular weight cut-off of 1KD and 10KD respectively to obtain purified Astragalus polysaccharides with molecular weight of 103～104The molecular weight of the middle molecular weight segment of astragalus polysaccharide between daltons is detected to be 103～104The relative content of the polysaccharide is 90 percent.
The main experimental reagents are Tris Base (Sigma), glycine (Sigma), protein marker (thermo), ZO-1Antibody (Affinity Biosciences), Anti-beta-actin Rabbit polyclonal Antibody (Affinity Biosciences), HRP Goat Anti-Rabbit IgG Antibody (Abcam), PVDF membrane (Millipore), ethanol (Tianjin Kohmu), xylene (Tianjin Kohmu), diethyl ether (Tianjin Fuyuyuu chemical industry), hematoxylin (Beijing Sorbabao), eosin (Beijing Sorbabao), BCA protein quantification kit (Biyun), SIGA Elisa kit (Shanghai enzyme remote biology), IFN-gamma Elisa kit (Shanghai enzyme remote biology), IL-4Elisa kit (Shanghai enzyme remote biology), IL-2 isa ET kit (enzyme remote biology), enzyme remote kit (enzyme remote biology gel), doctor-preparing kit (Risk protein gel), and Western doctor PA (Jinghai protein lysis), Western blot PA (Jinghai protein lysis PA) Tween-20 (the group of national medicine), cyclophosphamide (a leaf organism of Shanghai origin), a fecal DNA extraction kit (Tiangen Biochemical technology), a nucleic acid dye Gene Green (Tiangen Biochemical technology), a 2000bp DNA Marker (Tiangen Biochemical technology), a Loading buffer (Tiangen Biochemical technology), an acetic acid standard (Tianjin Kemiou), a propionic acid standard (Tianjin Kemiou), and a butyric acid standard (Tianjin Kemiou).
3.2 Experimental animals
Female Kunming mice, 18g +/-2 g, purchased from the fourth university of military medical science experimental animals center, and the animal production license number SYXK (shan) 2014-001.
3.3 Experimental animal grouping and handling
Female Kunming mice, weighing 18g +/-2 g, are suitable for breeding for one week, and are randomly divided into 12 mice in layers and numbered. The method comprises the steps of dividing the animals into a normal control group, a model group, a positive medicine group, an Astragalus Polysaccharide (APS) high-dose group and a astragalus polysaccharide (DAPS) low-dose group, a medium-molecular-weight astragalus polysaccharide (DAPS) high-dose group and a medium-molecular-weight astragalus polysaccharide (DAPS) low-dose group, and an intraperitoneal cyclophosphamide injection induced immunosuppression animal model, wherein the treatment methods of the animals in each group are shown in the table 8. Mouse body weights were recorded every two days. The administration was continued for 30 days, and after the drug treatment was completed, the mouse feces were aseptically collected. Then fasting the mice for 12 hours without water prohibition, recording the weight of each mouse, taking blood from eyeballs of the mice, taking off cervical vertebrae of the mice for sacrifice, separating serum, taking thymus and spleen of the mice and weighing the thymus and spleen; intercepting small intestine jejunum section of animal about 20cm, washing the section of tissue with PBS, and recovering intestinal mucus; freezing and storing jejunum section tissues for later use; cutting an intact jejunum section of the animal by about 2 cm, soaking the animal in 4% paraformaldehyde, and fixing for subsequent slicing and observation.
TABLE 8 Experimental animal groups and dosing regimens
Note: control is normal control group; model is a Model group; control, positive control group; APS (100) Astragalus polysaccharides low dose group; APS (400) Astragalus polysaccharides high dose group; DAPS (100) is a low-dose group of medium-molecular-weight fraction of Astragalus polysaccharides; DAPS (400) high dose group of medium molecular weight fraction of Astragalus polysaccharides
3.4 Experimental test indexes and method
3.4.1 morphological Observation of intestinal mucosa of mouse
Taking the fixed jejunum of the mouse, dehydrating, transparentizing, waxing, embedding, slicing according to a conventional paraffin section making method, sealing after HE dyeing, observing under a microscope, and measuring and recording the length (V) of villus and the depth (C) of crypt of the jejunum.
3.4.2 Elisa method for detecting influence of astragalus polysaccharides and medium molecular weight astragalus polysaccharides on contents of mouse intestinal mucus SIgA, IFN-gamma and IL-4
According to the operation of the corresponding kit specification, the contents of SIgA, IFN-gamma and IL-4 in intestinal mucus of the mouse are respectively detected, and after the content of protein in the intestinal mucus is determined by using a BCA protein quantitative kit, the SIgA, IFN-gamma and IL-4 in unit protein are calculated.
3.4.3 detection of mouse intestinal tissue Claudin ZO-1-Western blot method
Extracting small intestine tissue protein: freezing and grinding 100mg mouse intestinal tissue into fine powder by using liquid nitrogen, adding 750 mu L of RIPA lysate (750 mu L lysate, 7.5 mu L PMSF (phenylmethylsulfonyl fluoride, protease inhibitor) within minutes before use, continuously grinding until the intestinal tissue is in a liquid state, sucking into an EP tube, centrifuging at 4 ℃ for 14000r/min, centrifuging for 5min, sucking supernatant, taking 10 mu L of supernatant for protein quantification, and subpackaging the rest supernatant into a plurality of EP tubes by taking 30 mu L as a unit, and freezing and storing at-80 ℃ for later use in a refrigerator.
The extracted intestinal tissue protein is subjected to polyacrylamide gel electrophoresis separation, then is subjected to film transfer, sealing, primary antibody reaction, secondary antibody reaction, luminescence reaction, development and fixation, and then is photographed, and a gel image processing system analyzes the molecular weight and the net optical density value of a target zone.
3.4.4 Effect of Astragalus polysaccharides, middle molecular weight segment Astragalus polysaccharides on mouse intestinal flora- -16SrRNA Miseq illumana sequencing method
Extracting genome DNA of mouse feces by using a feces DNA extraction kit, and designing a primer by using a region sequence of bacteria 16sDNAV3-V4, wherein the primer sequence is as follows: upstream: ACTCCTACGGGAGGCAGCA, downstream: GGACTACHVGGGTWTCTAAT are provided. PCR amplification was performed. The amplification conditions were: 95 ℃ for 3min (denaturation); 95 ℃,30 s (denaturation) - -55 ℃,30 s (annealing) - -72 ℃, 45s (extension); after 30 cycles, 10min at 72 ℃. The PCR amplification system and the amplification conditions were as follows:
TABLE 9 PCR amplification System composition
After the amplification is finished, purifying, quantifying and uniformly analyzing the PCR product; then Miseq illiminina sequencing (completed by Shanghai Mergi biomedicine science and technology Co., Ltd.) is carried out, and intestinal bacteria identification and related informatics analysis are carried out, so as to detect the diversity of intestinal flora and the change of community structure.
3.4.5 measurement of SCFA (short chain fatty acid) in mouse feces
200mg of mouse feces was sufficiently dissolved in 1.5mL of chromatographic ethanol, centrifuged (10000r/min, 5 min), allowed to stand at 4 ℃ for 30min, and the supernatant was aspirated by a 1mL syringe and filtered through a 0.22 μm filter into a gas vial for use. Chromatographic conditions are as follows: chromatography column AT-WaX (30 m.times.0.25 mm.times.0.25 μm); temperature program of chromatographic column: maintaining the initial temperature at 40 deg.C for 1 min; raising the temperature to 180 ℃ at the speed of 8 ℃/min, and keeping the temperature for 1 min; the temperature is raised to 200 ℃ at the rate of 20 ℃/min and kept for 5 min. Carrier gas: he; flow rate of carrier gas: 1.2 mL/min; sample inlet temperature: 200 ℃; and (3) sample introduction mode: no split flow; sample introduction amount: 1 mu L of the solution; detector temperature (FID): 230 ℃ to 230 ℃. And (3) taking acetic acid, propionic acid and butyric acid as standard substances, and carrying out quantitative analysis.
3.5 results of the experiment
3.5.1 Effect of Astragalus polysaccharides, middle molecular weight fraction of Astragalus polysaccharides on mouse intestinal mucosa morphology
The intestinal villi of animals (N.control) growing in the natural state are compact, neat and complete; CY treated mice (Model) had shorter, thicker, swollen, atrophic, ruptured intestinal villi; after the treatment of the Lizhu Changle (P.control), the astragalus polysaccharide and the astragalus polysaccharide with medium molecular weight segment, the length of villus in the small intestine has obvious recovery trend, swelling, atrophy and fracture phenomena are reduced or even disappeared in different degrees, and the arrangement structure of the villus is also recovered to be regular.
The effects of astragalus polysaccharides and medium molecular weight astragalus polysaccharides on the length (V), crypt depth (C) and V/C of mouse small intestine villi are shown in Table 10. The results show that the V value of the model group is reduced, and the difference is significant compared with the normal group. The V value can be improved by each drug treatment group, and the data difference has obvious significance. Compared with the normal group, the model group C is higher, the value of each drug treatment group C is lower than that of the model group, and the differences except the astragalus polysaccharide (100 mg/kg) group have significant meaning. Compared with the normal group, the V/C value of the model group is reduced, the V/C value can be improved by treating each drug group, and the data difference has significant meaning.
TABLE 10 Effect of APS and DAPS on immunosuppressed mouse Small intestine V, C, V/C
Control is normal control group; model is a Model group; control, positive control group; DAPS (mg/kg) of a medium molecular weight fraction Astragalus polysaccharides; APS (mg/kg) Astragalus polysaccharides group
Note: compared with the normal group, # p < 0.05, # p < 0.01; p < 0.05, p < 0.01, compared to model group. Note, shared with normal control, # p < 0.05, # p < 0.01; compared with model control, p < 0.05, p < 0.01.
3.5.2 Effect of Astragalus polysaccharides, DAPS on mouse intestinal mucus SIGA, IFN-Gamma, IL-4 content
As shown in table 11, the SIgA secretion amount in intestinal mucus of the model group was significantly decreased, and the SIgA secretion amounts of the other groups were higher than that of the model group, and the data difference was significant, and compared with APS (400), the DAPS (400) intestinal mucus had a higher SIgA content and the difference was significant. In FIG. 2, # p < 0.05 compared with the normal group; p < 0.01, compared to model group; compared with astragalus polysaccharide (400), delta p is less than 0.05.
TABLE 11 Effect of APS and DAPS on the amount of SIgA in intestinal mucus from immunosuppressed mice
Such as
As shown in Table 12, the IFN-gamma/IL-4 values of the mice in the model group are remarkably reduced, the IFN-gamma/IL-4 values of the rest groups are recovered, the data difference is significant, and compared with APS (400), the IFN-gamma/IL-4 values in intestinal mucus of DAPS (400) are higher, and the difference is significant.
TABLE 12 Effect of APS and DAPS on immunosuppression of IFN-. gamma./IL-4 in intestinal mucus of mice
As shown in figure 1, the intestinal mucosa mechanical barrier of the immunosuppressed mice is damaged, the expression of zonulin ZO-1 in small intestinal tissues is obviously reduced, and after the treatment of the drugs, the expression of the zonulin is recovered, wherein the recovery effect of the astragalus polysaccharide in the medium molecular weight section is better than that of APS. In FIG. 1, N.control is a normal control group; model is a Model group; control, positive control group; DAPS is a middle molecular weight astragalus polysaccharide group; APS Astragalus polysaccharides group
Note: compared with the normal group, # # p is less than 0.01; p < 0.05, p < 0.01; Δ p < 0.05 compared to APS (400); compared to APS (100), p < 0.05. n-3).
3.5.4 influence of Astragalus polysaccharides and middle molecular weight fraction of Astragalus polysaccharides on mouse intestinal flora
The analysis of the sample dilution curve data during the sequencing of the mouse intestinal flora 16SrRNA shows that the OUT level number is not increased along with the increase of the sequencing sequence number, the dilution curve tends to be flat, the sequencing data are reasonable, and the subsequent data analysis is meaningful. There are several indexes describing the structural diversity of intestinal flora, wherein Ace and Chao indexes represent the abundance of the flora, and Shannon and Simpson indexes represent the diversity of the flora. The larger the Ace index and the Chao index are, the higher the representation richness is; the larger the Shannon index is, the higher the community diversity is; the smaller the Simpson index, the higher the diversity of the representative community. From fig. 2, it can be derived: compared with Ncontrol, the abundance of Model (Ace, Chao) is reduced (p < 0.05), while the Chao indexes of Pcontrol, astragalus polysaccharide with middle molecular weight and astragalus polysaccharide are obviously increased (p < 0.05); the Ace index of Pcontrol and medium molecular weight section astragalus polysaccharide is obviously increased (p is less than 0.05); the Ace index of astragalus polysaccharide has a recovery trend, but has no significant difference. The community diversity index Shannon index change difference among groups is small, the Simpson index shows that the diversity of the model group has a descending trend, and only the astragalus polysaccharide group with the middle molecular weight section has a certain recovery effect on Simpson.
PCoA analysis (principal co-ordinates analysis) was used to study the similarity and differences in the composition of the intestinal flora in each group. The closer the relative distance between the numerical samples of the abscissa and ordinate axes in the PCoA chart is, the higher the similarity between the two groups is, otherwise, the greater the difference is. The percentage of the horizontal and vertical axes represents the proportion of different factors contributing to the variability between the different groups. FIG. 3 is a PCoA analysis chart showing the structure of intestinal bacteria among the groups, and it can be seen from the analysis chart that Model is far away from Ncontrol, Pcontrol, the distance between the medium molecular weight fraction of Astragalus polysaccharides and Ncontrol is relatively close, the recovery trend is relatively obvious, and the recovery trend of Astragalus polysaccharides is relatively weak.
Fecal DNA from all groups of samples was detected by Miseq illumina sequencing in the V3-V4 region, with a total of 8 major phyla detected, with less than 1% of phyla assigned to others. These eight phyla are, respectively, deferobacteres (deironium), spirochaetee (spirillum), Fusobacteria (fusobacterium), Proteobacteria (proteobacterium), Actinobacteria (actinomycetes), Firmicutes (Firmicutes), bactoidides (Bacteroidetes), unclassified _ k __ noran. Statistics were taken of the Firmicutes/bacterioides changes in each group, as shown in Table 13. The Firmicutes/bacterioides ratio in the Model is very significantly increased compared to Ncontrol (p < 0.01); compared with a Model, the proportion of Firmicutes/bacterioides in Pcontrol and DAPS is remarkably or extremely remarkably reduced (p is less than 0.05, p is less than 0.01), the recovery trend is obvious, and the proportion of APS is not reduced; DAPS decreased significantly compared to APS (p < 0.05).
TABLE 13 Effect of APS and DAPS on Firmicutes/bacteriodes
APS, Astragalus polysaccharides group; DAPS is a middle molecular weight astragalus polysaccharide group; ncontrol, normal control group; model is a Model group; pcontrol positive control group
Note: compared with the normal group, # # p is less than 0.01; p < 0.05, p < 0.01; Δ p < 0.05 compared to APS.
Analysis at the genus level showed that less than 1% of the genera were assigned to others, with greater than 1% of the 35 genera detected in total. Table 14 gives statistics on the genera with significant variation among the groups. The major changes in relative contents in the Model compared to Ncontrol are: the relative abundance of Bacteroides, Lactobacillus, prevotellaceae _ UGG-001, norak _ f _ Bacteroidales _ S24-7_ group, Odoribacter, Lachnospiraceae _ NK4A136_ group, Alloprovella, etc. decreases (p < 0.05 or p < 0.01), and the relative abundance of Rhodococcus increases (p < 0.05). Compared with a Model, the Pcontrol and DAPS genera have obvious recovery tendency (p is less than 0.05 or p is less than 0.01), the Lactobacillus recovery effect in the APS group is obvious (p is less than 0.05), and the recovery tendency to other genera is not obvious. Compared with APS, Bacteroides, prevotellaceae _ UGG-001, norak _ f _ Bacteroides _ S24-7_ group, Odoribacter, Lachnospiraceae _ NK4A136_ group, Allopreviella in DAPS are increased remarkably or extremely remarkably (p < 0.05 or p < 0.01), and Rhodococcus is decreased remarkably (p < 0.05). In conclusion, the DAPS has obvious recovery effect on the disturbance of the level of the intestinal flora, and the effect is better than that of the APS.
TABLE 14 Effect of APS and DAPS on genus levels
APS, Astragalus polysaccharides group; DAPS is a middle molecular weight astragalus polysaccharide group; ncontrol, normal control group; model is a Model group; pcontrol is positive control group; compared with the normal group, # p < 0.05, # p < 0.01; p < 0.05, p < 0.01; Δ p < 0.05 and Δ p < 0.01, compared to APS.
3.5.5 measurement of SCFA (short chain fatty acid) in mouse feces
Respectively injecting samples by using standard acetic acid, propionic acid and butyric acid, wherein standard curves are respectively as follows: 0.00000418 x +0.52159094 for y, 0.999 for R2 (acetic acid); y 0.00000013x +0.02089694, R2 0.999 (propionic acid); y is 0.00000011x +0.01671176 and R2 is 0.999 (butyric acid).
Short Chain Fatty Acids (SCFA) are fermentation products of intestinal flora with carbohydrates as substrates, mainly comprising acetic acid, propionic acid, butyric acid, valeric acid, etc. The relative content of SCFA in each group was determined from the calibration curve. As shown in fig. 4, compared with n.control, the content of acetic acid (acetic acid), propionic acid (propionic acid) and butyric acid (butyrate) in the feces of CY-induced immunosuppressed mice showed a significantly decreased trend (p < 0.05). APS in FIG. 4, Astragalus polysaccharides group; DAPS is a middle molecular weight astragalus polysaccharide group; control is normal control group; model is a Model group; control, positive control group;
note: compared with the normal group, # p < 0.05, # p < 0.01; p < 0.05, p < 0.01; Δ p < 0.05 compared to APS (400).
After the astragalus polysaccharide and the medium molecular weight astragalus polysaccharide are treated, the content of short-chain fatty acid in the excrement is increased, and compared with a Model, the data difference has a significant meaning (p is less than 0.05). Therefore, the astragalus polysaccharide and the medium molecular weight section of astragalus polysaccharide can recover the reduction of the immunosuppressed mouse intestinal flora metabolite SCFA caused by CY, and the result shows that the recovery effect of the medium molecular weight section of astragalus polysaccharide is better than that of the astragalus polysaccharide.
3.6 conclusion
The astragalus polysaccharide has the function of promoting immunity; the astragalus polysaccharide also has certain influence on intestinal flora disorder of immunized mice induced by cyclophosphamide, and can promote the generation of intestinal bacteria metabolite short-chain fatty acid; therefore, the influence of astragalus polysaccharides on the mucosal immune function is related to the influence of astragalus polysaccharides on the microecology of intestinal flora; in vitro intestinal culture experiments prove that the astragalus polysaccharide can promote the secretion of SIgA and enhance the intestinal mucosa immune function. In multiple indexes detected, such as SIgA secretion, IFN-gamma/IL-4, small intestine tissue tight junction protein ZO-1 expression, intestinal microecological change, SCFA content spleen index and the like, the recovery effect of the astragalus polysaccharide with a medium molecular weight segment on immunosuppressive mice is superior to that of the astragalus polysaccharide, so that the astragalus polysaccharide hydrolysate has a better effect of promoting mucosal immunity.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. An application of middle molecular weight segment astragalus polysaccharide in preparing medicine or food for improving intestinal mucosa immunity and intestinal microecosystem function is characterized in that the middle molecular weight segment astragalus polysaccharide is obtained by hydrolyzing astragalus crude polysaccharide, and the molecular weight range of the middle molecular weight segment astragalus polysaccharide is 103-104Dalton;
the preparation method of the astragalus polysaccharide with the medium molecular weight segment comprises the steps of dissolving the crude astragalus polysaccharide in hot water, adding acid, heating in a water bath, adding alkali liquor to adjust the pH value to be neutral, concentrating and drying the hydrolysate, and crushing and sieving to obtain an astragalus polysaccharide hydrolysate, wherein the content of the astragalus polysaccharide with the medium molecular weight segment in the astragalus polysaccharide hydrolysate is not less than 50%;
the acid is hydrochloric acid, the concentration of the hydrochloric acid in the astragalus crude polysaccharide solution is adjusted to be 0.16mol/L, the water bath heating temperature is 80 ℃, and the time is 5 hours.
2. The use of claim 1, wherein the hydrolysate is further subjected to dialysis treatment after concentration and before drying to obtain purified medium molecular weight fraction astragalus polysaccharides.
3. The use of claim 1, wherein the crude astragalus polysaccharides are extracted by the following method: pulverizing radix astragali, extracting with warm water, concentrating the extractive solution, removing protein, centrifuging, concentrating the supernatant, washing with 80% ethanol, and precipitating to obtain radix astragali crude polysaccharide.
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