CN110218265A - A kind of middle-molecular-weihydroxyethyl section astragalus polyose, preparation method and applications - Google Patents
A kind of middle-molecular-weihydroxyethyl section astragalus polyose, preparation method and applications Download PDFInfo
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- CN110218265A CN110218265A CN201910561282.5A CN201910561282A CN110218265A CN 110218265 A CN110218265 A CN 110218265A CN 201910561282 A CN201910561282 A CN 201910561282A CN 110218265 A CN110218265 A CN 110218265A
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- astragalus polyose
- molecular
- weihydroxyethyl
- section
- acid
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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
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- A—HUMAN NECESSITIES
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a kind of middle-molecular-weihydroxyethyl section astragalus polyoses, preparation method and applications, belong to biological medicine manufacturing technology field.Middle-molecular-weihydroxyethyl section astragalus polyose of the invention is hydrolyzed by Radix Astragali Thick many candies and is obtained, and the molecular weight ranges of the middle-molecular-weihydroxyethyl section astragalus polyose are 103‑104Dalton, the preparation method the invention also discloses middle-molecular-weihydroxyethyl section astragalus polyose are to carry out sour water solution to Radix Astragali Thick many candies, obtain the middle-molecular-weihydroxyethyl section astragalus polyose.The invention also discloses middle-molecular-weihydroxyethyl section astragalus polyoses to prepare the application in drug or food for improving Mucosal Immunity and intestinal microecology system function.The middle-molecular-weihydroxyethyl section astragalus polyose generated after astragalus polyose hydrolysis of the invention is more preferable than natural astragalus polyose effect in adjusting Mucosal Immunity and intestinal microecology system aspects, enhances the health-care effect of astragalus polyose.Astragalus polyose acid hydrolysis process in the present invention is simple, it is easy to accomplish industrialized production.
Description
Technical field
The present invention relates to biological medicine manufacturing technology fields, and in particular to a kind of preparation side of middle-molecular-weihydroxyethyl section astragalus polyose
Method and its application.
Background technique
Radix Astragali is one of large Chinese medicine that tcm clinical practice is most widely used, since Radix Astragali is curative for effect, the scope of application
Extensively, it is praised highly by ancient Chinese medicine doctor.Radix Astragali is sweet in flavor and warm in property, and there is tonifying Qi and lifting yang, invigorating qi for consolidating superficies, inducing diuresis for removing edema and torr sore myogenic etc. to make
With；Clinically there is preferable treatment to the circulatory system, nervous system, digestive system, respiratory system, endocrine and disease in the blood system
Effect.Pharmaceutical research shows that Astragalus Root P.E or monomeric compound are protected in anti-inflammatory, immunological regulation, antitumor, myocardial ischemia
Shield, diabetes, anti-oxidant, anti-aging etc. has specific effect.From the point of view of modern study, the primary efficacy of Radix Astragali
It is to adjust immune function, Radix Astragali acts on close phase with " invigorating qi for consolidating superficies " in its tradition application to the adjustment effect of immune function
Close, once said in " The Yellow Emperor's Canon of Internal Medicine Ling Shu Miraculous Pivot or Divine Axis ": " defending gas not seek, perverse trend occupies it ", explicitly pointing out " defending gas " is that body is interior with perverse trend phase
A kind of defense mechanism contended with, modern research suggests that defending, the deficiency of vital energy is weak to be mainly shown as immunologic hypofunction, therefore, the core of Radix Astragali
Effect is the influence to immune function.Having numerous studies in the prior art confirms that astragalus polyose is the main active component of Radix Astragali
One of.Natural astragalus polyose molecular weight is higher, and average molecular weight cannot be digested and assimilated, therefore 100,000 or more by human normal
The application proposes a kind of middle-molecular-weihydroxyethyl section astragalus polyose, preparation method and applications.
Summary of the invention
The purpose of the invention is to overcome the problems of the prior art, a kind of middle-molecular-weihydroxyethyl section astragalus polyose, system are provided
Preparation Method and its application.
An object of the present invention is to provide a kind of middle-molecular-weihydroxyethyl section astragalus polyose, is hydrolyzed and is obtained by Radix Astragali Thick many candies, should
The molecular weight ranges of middle-molecular-weihydroxyethyl section astragalus polyose are 103-104Dalton.
The second object of the present invention provides a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose, specific as follows: first will be yellow
Then stilbene Thick many candies hot water dissolving, acid adding and heating water bath are added lye and adjust pH to neutrality, after hydrolyzate concentrate drying,
It pulverizes and sieves, obtains astragalus polyose hydrolysate, the content of middle-molecular-weihydroxyethyl section astragalus polyose is not less than in the astragalus polyose hydrolysate
50%.
Preferably, also having carried out dialysis treatment before dry after hydrolyzate concentration, the middle-molecular-weihydroxyethyl section obtained after purification is yellow
Astragalus polysaccharides.
Preferably, acid is the organic acid and inorganic acid for capableing of hydrolyzing glucosidic bonds.
Preferably, acid is hydrochloric acid, being adjusted to concentration of hydrochloric acid in Radix Astragali Thick many candies solution is 0.03~0.30mol/L, and water-bath adds
Hot temperature is 10~100 DEG C, and the time is 1~8h.
Preferably, acid is trifluoroacetic acid, being adjusted to trifluoroacetic acid concentration in Radix Astragali Thick many candies solution is 0.05~0.30mol/
L's, water bath heating temperature is 10~100 DEG C, and the time is 1~8h.
Preferably, the extracting method of the Radix Astragali Thick many candies is as follows: water temperature extraction takes after Milkvetch Root is crushed, and concentration mentions
Liquid, removing protein are taken, centrifugation precipitates through 80% ethanol washing after supernatant concentration, obtains Radix Astragali Thick many candies.
The third object of the present invention be above-mentioned middle-molecular-weihydroxyethyl section astragalus polyose preparation for improve Mucosal Immunity and
The drug of intestinal microecology system function or the application in food.
Compared with prior art, the beneficial effects of the present invention are: in the present invention middle-molecular-weihydroxyethyl section astragalus polyose molecular weight
Range is 103~104Between dalton, molecular diffusion rates are very fast, are easy to be utilized by enterobacteriaceae, are also easier to thin with intestinal mucosa
Cell phase interaction can preferably realize the function of adjusting intestinal mucosal immune and intestinal microecology system；
The present invention hydrolyzes natural astragalus polyose to obtain the astragalus polyose of middle-molecular-weihydroxyethyl section, and uses acid-hydrolyzed method
Astragalus polyose can be hydrolyzed to the astragalus polyose of middle-molecular-weihydroxyethyl section, and be unlikely to thoroughly for astragalus polyose to be degraded to monosaccharide or widow
Sugar, middle-molecular-weihydroxyethyl section astragalus polyose content is not less than in the astragalus polyose hydrolysate obtained through acid hydrolysis process of the invention
50%；
The present invention has also been experimentally confirmed the middle-molecular-weihydroxyethyl section astragalus polyose generated after astragalus polyose hydrolysis of the invention
It is more preferable than natural astragalus polyose effect in adjusting Mucosal Immunity and intestinal microecology system aspects, enhance the guarantor of astragalus polyose
Strong effect.Astragalus polyose acid hydrolysis process in the present invention is simple, it is easy to accomplish industrialized production.
Detailed description of the invention
Fig. 1 be APS of the present invention and DAPS handle to small intestine, immunosuppressed mice small intestine ZO-1 influence (N=3)；
Fig. 2 is mouse intestinal flora microecosystem diversity indices of the present invention and diversity index (n=3)；
Fig. 2 a is the Chao index of mouse intestinal flora after APS, DAPS of the present invention processing；
Fig. 2 b is the Ace index of mouse intestinal flora after APS, DAPS of the present invention processing；
Fig. 2 c is the Shannon index of mouse intestinal flora after APS, DAPS of the present invention processing；
Fig. 2 d is the Simpson index of mouse intestinal flora after APS, DAPS of the present invention processing；
Fig. 3 is the PCoA analysis chart (n=3) of mouse intestinal flora after APS, DAPS of the present invention processing；
Fig. 4 be APS, DAPS of the present invention handle after to mouse intestinal flora metabolite SCFA influence (N=
5)。
Fig. 4 a is the influence after APS, DAPS are handled to mouse intestinal flora metabolite acetic acid.
Fig. 4 b is the influence after APS, DAPS are handled to mouse intestinal flora metabolite propionic acid.
Fig. 4 c is the influence after APS, DAPS are handled to mouse intestinal flora metabolite butyric acid.
Specific embodiment
With reference to the accompanying drawing and embodiment specific embodiments of the present invention will be described in detail, it is to be understood that this
The protection scope of invention is not limited by the specific implementation.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Embodiment 1
By prepare be 1000, middle-molecular-weihydroxyethyl section astragalus polyose tablet containing active constituent for raw materials used and auxiliary material and its
Quality proportioning is as follows:
1, the extraction of astragalus polyose: water temperature extraction takes after Milkvetch Root is crushed, and concentrated extracting solution is removed by Sevag method
Albumen, centrifugation, after supernatant concentration, 80% ethanol precipitation, washing of precipitate obtains Radix Astragali Thick many candies；
2, the sour water solution of Radix Astragali Thick many candies: after Radix Astragali Thick many candies hot water dissolving, it is 0.16mol/L that hydrochloric acid to concentration, which is added,
(mass percent concentration 0.05%w/w) is then added equivalent NaOH solution and adjusts pH to neutrality in 80 DEG C of water-bath 3h,
Hydrolyzate is concentrated into medicinal extract shape, and crushed after being dried, sieving are obtained rich in molecular weight between 103~104Middle molecule between dalton
Measure section astragalus polyose product.
By prepare be 1000, tablet of middle-molecular-weihydroxyethyl section astragalus polyose containing active constituent for raw materials used and auxiliary material and
Its quality proportioning is as follows: being rich in middle-molecular-weihydroxyethyl section astragalus polyose product 150g, cornstarch 1450g, magnesium stearate 5g press preparation
The common process for learning tablet carries out, every slice weight 0.3g, the every astragalus polyose of section containing middle-molecular-weihydroxyethyl 150mg.Usage and dosage: it is oral,
3 tablets once, and three times per day, children are cut down according to the circumstance.
Embodiment 2
By prepare containing active constituent be middle-molecular-weihydroxyethyl section astragalus polysaccharide particles agent 1000 wrap for raw materials used and auxiliary material and
Its quality proportioning is as follows:
1, the extraction of astragalus polyose: water temperature extraction takes after Milkvetch Root is crushed, and concentrated extracting solution is removed by Sevag method
Albumen, centrifugation, after supernatant concentration, 80% ethanol precipitation, washing of precipitate obtains Radix Astragali Thick many candies；
2, the sour water solution of Radix Astragali Thick many candies: after Radix Astragali Thick many candies hot water dissolving, addition trifluoroacetic acid is adjusted to concentration and is
0.1mol/L (0.8%w/w) is in 80 DEG C of water-bath 3h；In 80 DEG C of water-bath 3h, equivalent NaOH solution is then added and adjusts pH into
Property, it is respectively 1KD and the bag filter dialysis treatment of 10KD with molecular cut off after hydrolyzate concentration, crushed after being dried, sieving
The molecular weight of purifying is obtained between 103~104Middle-molecular-weihydroxyethyl section astragalus polyose product between dalton.
The raw materials used and auxiliary material for preparing and be wrapped containing the granule 1000 that active constituent is middle-molecular-weihydroxyethyl section astragalus polyose
And its quality proportioning is as follows: middle-molecular-weihydroxyethyl section astragalus polyose product 450g, cane sugar powder 2000g, dextrin 650g.By galenic pharmacy particle
The common process of agent carries out, every packet weight 3g, the astragalus polyose of section containing middle-molecular-weihydroxyethyl 450mg.Usage and dosage: it is oral, one bag every time, one
It is three times.
Embodiment 3
By prepare containing active constituent be middle-molecular-weihydroxyethyl section astragalus polyose capsule 1000 for raw materials used and auxiliary material and
Its quality proportioning is as follows:
1, the extraction of astragalus polyose: water temperature extraction takes after Milkvetch Root is crushed, and concentrated extracting solution is removed by Sevag method
Albumen, centrifugation, after supernatant concentration, 80% ethanol precipitation, washing of precipitate obtains Radix Astragali Thick many candies；
2, the sour water solution of Radix Astragali Thick many candies: after Radix Astragali Thick many candies hot water dissolving, it is 0.16mol/ that hydrochloric acid, which is added, and is adjusted to concentration
Then L (0.5%), 80 DEG C of water-bath 6h are added equivalent NaOH solution and adjust pH to neutrality, hydrolyzate is concentrated into medicinal extract shape, does
It is crushed after dry, sieving is obtained rich in molecular weight between 103~104Middle-molecular-weihydroxyethyl section astragalus polyose product between dalton.
The raw materials used and auxiliary material for preparing and be wrapped containing the granule 1000 that active constituent is middle-molecular-weihydroxyethyl section astragalus polyose
And its quality proportioning is as follows: middle-molecular-weihydroxyethyl section astragalus polyose product 150g, cornstarch 150g.By the routine of galenic pharmacy capsule
Technique carries out, every weight 3g, the astragalus polyose of section containing middle-molecular-weihydroxyethyl 150mg.Usage and dosage: it is oral, 2 tablets each time, three times a day.
Embodiment 4
By prepare containing active constituent be middle-molecular-weihydroxyethyl section astragalus polysaccharides oral liquid 1000 for raw materials used and auxiliary material and
Its quality proportioning is as follows:
1, the extraction of astragalus polyose: water temperature extraction takes after Milkvetch Root is crushed, and concentrated extracting solution is removed by Sevag method
Albumen, centrifugation, after supernatant concentration, 80% ethanol precipitation, washing of precipitate obtains Radix Astragali Thick many candies；2, the acid of Radix Astragali Thick many candies
Hydrolysis: after Radix Astragali Thick many candies hot water dissolving, be added trifluoroacetic acid be adjusted to concentration be 0.10mol/L (0.5%), 60 DEG C of water-bath 6h,
Then equivalent NaOH solution is added and adjusts pH to neutrality, is respectively 1KD and 10KD with molecular cut off after hydrolyzate concentration
Bag filter dialysis treatment, crushed after being dried, sieving obtain the molecular weight of purifying between 103~104Middle-molecular-weihydroxyethyl between dalton
Section astragalus polyose product.
The raw materials used and auxiliary material for preparing containing oral solution 1000 that active constituent is middle-molecular-weihydroxyethyl section astragalus polyose
And its quality proportioning is as follows: middle-molecular-weihydroxyethyl section astragalus polyose 450g, cane sugar powder 6000g, benzoic acid 10g, distilled water 10000ml.
It is carried out by the common process of galenic pharmacy oral solution, every 10ml, the astragalus polyose of section containing middle-molecular-weihydroxyethyl 450mg.Usage and dosage: mouth
Clothes, 1 every time, three times a day.
Embodiment 5
Its process is as follows for preparing containing the bread that active constituent is middle-molecular-weihydroxyethyl section astragalus polyose:
1, the extraction of astragalus polyose: water temperature extraction takes after Milkvetch Root is crushed, and concentrated extracting solution is removed by Sevag method
Albumen, centrifugation, after supernatant concentration, 80% ethanol precipitation, washing of precipitate obtains Radix Astragali Thick many candies；
2, the sour water solution of Radix Astragali Thick many candies: after Radix Astragali Thick many candies hot water dissolving, it is 0.16mol/L that hydrochloric acid to concentration, which is added,
(mass percent concentration 0.05%w/w) is then added equivalent NaOH solution and adjusts pH to neutrality in 80 DEG C of water-bath 3h,
It is respectively 1KD and the bag filter dialysis treatment of 10KD with molecular cut off after hydrolyzate concentration, crushed after being dried, sieving obtain
The molecular weight of purifying is between 103~104Middle-molecular-weihydroxyethyl section astragalus polyose product between dalton.
Raw materials used and auxiliary material and its quality for preparing containing the bread that active constituent is middle-molecular-weihydroxyethyl section astragalus polyose
It matches as follows: middle-molecular-weihydroxyethyl section astragalus polyose product 5.4g, cane sugar powder 930g flour, vegetable oil 50g, active dry yeasr 20g.It presses
More solito breadmaking process makes bread.Bread 250g, the astragalus polyose of section containing middle-molecular-weihydroxyethyl 1.35g are eaten for each person every day.
In the food such as such as biscuit, cake, middle-molecular-weihydroxyethyl section astragalus polyose can also be added, by middle-molecular-weihydroxyethyl for each person every day
Section 1~2g of astragalus polyose amount is advisable.
We demonstrate the stability problem of the hydrolysis effect of astragalus polyose and hydrolysis process under different hydrolysising conditions；In order to
Middle-molecular-weihydroxyethyl section astragalus polyose is verified in the helpfulness for adjusting Gut mucosal immune function and intestinal microecology system aspects, applicant
Also done various technological parameters to when pharmacodynamic test, related experiment is as follows:
One, the acid hydrolysis process research of astragalus polyose:
The preparation of 1.1 astragalus polyoses
Radix Astragali dry powder 100g is taken, by solid-liquid ratio 1: 10 plus water refluxing extraction, filtering is collected filtrate, is repeated 3 times, and merges filter
Liquid, centrifuging and taking supernatant, supernatant are concentrated under reduced pressure into 400ml, and ethyl alcohol, which is then added, makes determining alcohol 80%, stand filtering for 24 hours,
It must precipitate, dry thick astragalus polyose i.e., it is dry to get astragalus polyose after thick astragalus polyose Sevage method takes off albumen processing.
It is detected through Coomassie Brilliant Blue, protein content < 1% in the astragalus polyose of preparation is detected through phend-sulphuric acid, the Radix Astragali of preparation
Sugared content is 57.40% (0.5740 ± 0.0174, n=5) in polysaccharide.
The detection of 1.2 polysaccharide molecular weights --- High Performance Gel Permeation Chromatography:
1.2.1 chromatographic condition: Reprosil 125SEC chromatographic column, 5 μm, 300 × 7.8mm, 30 DEG C of column temperature, mobile phase is
Deionized water, flow velocity 0.5mL/min, detector be ELSD3300 evaporative light scattering detector (Alltech), 100 DEG C of temperature.
Dextran molecule amount standard items (Mw4320, Mw12600, Mw70800, Mw126000, Beijing Suo Laibao Science and Technology Ltd).
It should be noted that the range that efficiently separates of Reprosil 125SEC chromatographic column is 3000~500000, so meter
There is any discrepancy for molecular weight and the actual molecular weight meeting calculated, it is contemplated that the research purpose of this experiment, the splitter separating ranges are
It is enough to judge the hydrolysis situation of astragalus polyose and middle-molecular-weihydroxyethyl section astragalus polyose yield issues.
1.2.2 the production of molecular mass standard curve
Under above-mentioned chromatographic condition, respectively with dextran molecule amount standard items (Mw4320,12600,70800,126000,
Beijing Suo Laibao Science and Technology Ltd) and glucose sample detection.With retention time (min) for abscissa, relative molecular weight pair
Number (lg Mw) is that ordinate makees canonical plotting.
1.2.3 molecular weight analyte detects
Under above-mentioned chromatographic condition, sample is dissolved in deionized water sample detection.Bring sample retention time into standard curve
Equation in, calculate sample relative molecular mass (Mw).
The research of 1.3 astragalus polyose acid hydrolysis process
1.3.1 astragalus polyose hydrochloric acid hydrolysis process
10mg astragalus polyose is weighed, is put into tool plug test tube, investigates the influence of time and acid concentration respectively.
According to preliminary result, concentration of hydrochloric acid is respectively set are as follows: 0.08,0.160,0.320mol/L；Hydrolysis temperature is 80
DEG C, hydrolysis time 5h, by solution NaOH solution tune pH to neutrality after hydrolysis, after freeze-drying, with deionized water dissolving,
HPLC-ELSD analyzes its molecular weight information.
1.3.2 astragalus polyose trifluoroacetic acid hydrolysis technique
10mg astragalus polyose is weighed, is put into tool plug test tube, investigates the influence of time and acid concentration respectively.
According to preliminary result, trifluoroacetic acid concentration is respectively set are as follows: 0.05,0.10,0.20mol/L；Hydrolysis temperature is
80 DEG C, hydrolysis time 5h, by solution NaOH solution tune pH to neutrality after hydrolysis, after freeze-drying, with deionized water dissolving,
HPLC-ELSD analyzes its molecular weight information.
1.4 astragalus polyose acid hydrolysis process results of study
1.4.1 polysaccharide molecular weight standard curve
Using the glucan mark product and glucose of different relative molecular masses, according to 1.2 methods.Pass through HPSEC-ELSD
Method measures it in chromatographic column retention time, and with the retention time (t) of each mark product for X axis, lgMw is y-axis, draws standard curve
Regression equation are as follows: y=-0.281x+8.279, R2=0.9839.
The retention time of 1 various criterion dextran molecule of table
1.4.2 astragalus polyose hydrochloric acid hydrolyzes process results
After different concentration of hydrochloric acid hydrolysis, the molecular weight composition of polysaccharide in hydrolysate is measured, as a result as follows:
The hydrolysis effect of astragalus polyose under the conditions of the different concentration of hydrochloric acid of table 2
According to experimental result as can be seen that astragalus polyose is mainly by high, medium and low three parts different molecular weight section after hydrolysis
Material composition, wherein high molecular weight section is mainly unhydrolysed astragalus polyose, average relative molecular mass 1.5 × 105Dalton
Left and right, middle-molecular-weihydroxyethyl section are mainly made of the astragalus polyose of partial hydrolysis, and molecular weight is located at 103~104Between dalton, and
Low molecular weight section predominantly hydrolyzes more thorough monosaccharide or oligosaccharides composition, and molecular weight is less than 103Dalton.It can be with from result
Find out, with being gradually increasing for concentration of hydrochloric acid, high molecular weight section proportion is gradually reduced, middle low molecular weight section proportion by
Gradually rise, when concentration of hydrochloric acid is 0.16mol/L, the astragalus polyose content of high molecular weight section is reduced to 10% or so, and in point
The polysaccharide relative scale of son amount section reaches 70% or more, illustrates that most astragalus polyoses have all hydrolyzed, main in product
For the astragalus polyose of intermediate molecular weight size.When concentration of hydrochloric acid is 0.32mol/L, the substance relative scale of low molecular weight section reaches
To 90% or more, molecular weight illustrates astragalus polyose hydrolysis than more thoroughly between 400-500 dalton.
So hydrolysis temperature is 80 DEG C when selecting concentration of hydrochloric acid as 0.16mol/L, hydrolysis time is 5h as in preparation points
The appropriate hydrolysising condition of son amount section astragalus polyose.
1.4.3 astragalus polyose trifluoroacetic acid hydrolysis process results
After different trifluoroacetic acid concentration hydrolysis, the molecular weight composition of polysaccharide in hydrolysate is measured, as a result as follows:
The hydrolysis effect of astragalus polyose under the conditions of the different trifluoroacetic acid concentrations of table 3
Similar with the experimental result of hydrochloric acid hydrolysis, astragalus polyose is mainly by high, medium and low three parts different molecular weight after hydrolysis
The material composition of section, wherein high molecular weight section is mainly unhydrolysed astragalus polyose, average relative molecular mass 1.5 × 105Road
Er Dun or so, middle-molecular-weihydroxyethyl section are mainly made of the astragalus polyose of partial hydrolysis, and molecular weight is located at 103~104Dalton it
Between, and low molecular weight section predominantly hydrolyzes more thorough monosaccharide or oligosaccharides composition, molecular weight is lower than 1000 dalton.From knot
Fruit can be seen that being gradually increasing with trifluoroacetic acid concentration, and high molecular weight section proportion is gradually reduced, middle low molecular weight section
Proportion be gradually increasing, according to experimental result as can be seen that trifluoroacetic acid concentration be 0.10mol/L when, high molecular weight
The astragalus polyose content of section is reduced to 25% or so, and the relative scale of middle-molecular-weihydroxyethyl section reaches 60% or more, illustrates most absolutely
Number astragalus polyose has all hydrolyzed, and relative molecular weight is respectively in 4000 dalton or so.It is in trifluoroacetic acid concentration
When 0.20mol/L, low molecular weight segment molecule amount is very high lower than 1000 relative scale in product, illustrates that hydrolysis has been compared
Thoroughly.
So hydrolysis temperature is 80 DEG C when selecting trifluoroacetic acid concentration as 0.10mol/L, hydrolysis time is 5h as system
The appropriate hydrolysising condition of standby middle-molecular-weihydroxyethyl section astragalus polyose.
1.4.4 conclusion:
It is 0.16mol/L that the hydrochloric acid of astragalus polyose, which hydrolyzes better suited concentration,；The appropriate acid concentration of trifluoroacetic acid is
In the above conditions, when different sour water solutions, temperature can use 80 DEG C to 0.10mol/L, and hydrolysis time can use 5h.
1.4.6 discussing
When astragalus polyose hydrolyzes ratio more thoroughly, product is mainly oligosaccharides and monosaccharide, and molecular weight is generally below 1000 dongles
, and oligosaccharides and monosaccharide substance are smaller to mucosal immunity function effect；Hydrolysate molecular weight is located at 103~104Dalton
Between polysaccharose substance may existing good mucosal immunity regulatory function, also have intestinal microecology system regulatory function, institute
Primary evaluation index with astragalus polyose hydrolysis process is exactly to improve hydrolysate middle-molecular-weihydroxyethyl as far as possible to be located at 103~104Dongle
Polysaccharose substance relative amount between.Under the conditions of more excellent in the above experiment, molecular weight is located at 103~104In dalton
Relative amount of the molecular weight section astragalus polyose in total hydrolysate is not less than 50%.
The main factor for influencing astragalus polyose hydrolysis is acid concentration, and acid concentration is excessively high, will lead to excessive hydrolysis, Nai Zhiquan
Portion is hydrolyzed to monosaccharide, and acid concentration is too low, and hydrolysis degree is too low, is located at 103~104Polysaccharose substance product between dalton
It is very few.The conditions such as hydrolysis time, hydrolysis temperature are relatively small on hydrolysis degree influence, can be according to the actual needs tune of production
It is whole.In addition, there are many type for the acid that the hydrolysis of astragalus polyose can choose, such as sulfuric acid, phosphoric acid, nitric acid, but theoretically
Analysis, sulfuric acid, nitric acid are oxidizing, and the type of hydrolysate can be relatively more, and the structure of product is it can also happen that variation, produces simultaneously
It is mixed with other particles such as sulfate radical, nitrate anion in object, influences the follow-up function of product, environmental pollution is also higher.And hydrochloric acid water
What solution technique introduced is chloride ion, and the boiling point of trifluoroacetic acid is 72.4 DEG C, and can volatilize removing in the drying process, in the product
Residual it is smaller to the function effect of future products, so in the factorial production, select hydrochloric acid, trifluoroacetic acid to have certain excellent
Gesture.
The stability study of the acid hydrolysis process of two astragalus polyoses
Compared with enzyme hydrolysis method, using acid-hydrolysis method Polysaccharides since hydrolytic sites randomness is high, product component is opposite
Complexity, so under the conditions of acid hydrolysis process, whether product composition stable to be an important factor for being related to product efficacy, so we
Explore the composition steadiness of acid hydrolysis products under the process conditions of optimization.For the present invention, acid hydrolysis products
Core requirement be astragalus polyose after hydrolysis, product be the lesser astragalus polyose of the lesser degree of polymerization of molecular weight, and be unlikely to by
Astragalus polyose is excessively hydrolyzed to monosaccharide or oligosaccharides and reduces its immunoloregulation function.By detecting astragalus polyose acid hydrolysis products
Molecular weight anabolic reaction hydrolysis process steadiness.
The preparation of 2.1 astragalus polyoses: with 1.1
The detection of 2.2 polysaccharide molecular weights --- High Performance Gel Permeation Chromatography: with 1.2
2.3 astragalus polyose acid hydrolysis process stability studies
2.3.1 astragalus polyose hydrochloric acid hydrolysis technology stability research
10mg astragalus polyose is weighed, is put into tool plug test tube, addition concentration of hydrochloric acid is 0.160mol/L；Hydrolysis temperature is 80
DEG C, hydrolysis time 5h, by solution NaOH solution tune pH to neutrality after hydrolysis, after freeze-drying, with deionized water dissolving,
HPLC-ELSD analyzes its molecular weight information.In triplicate, hydrolysate molecular weight distribution compares between calculating different batches for experiment
Relative standard deviation (RSD) variation of example.
2.3.2 astragalus polyose trifluoroacetic acid hydrolysis process stabilizing Journal of Sex Research
10mg astragalus polyose is weighed, is put into tool plug test tube, addition trifluoroacetic acid acid concentration is 0.10mol/L；Hydrolysis temperature
Degree is 80 DEG C, hydrolysis time 5h, by solution NaOH solution tune pH to neutrality after hydrolysis, after freeze-drying, uses deionized water
Dissolution, HPLC-ELSD analyze its molecular weight information.Experiment in triplicate, calculates hydrolysate molecular weight distribution between different batches
Relative standard deviation (RSD) variation.
2.4 result
2.4.1 it is measured in chromatographic column retention time, with the reservation of each mark product by HPSEC-ELSD method with 1.4.1
Time (t) is X-axis, and lgMw is y-axis, draws the regression equation of standard curve are as follows: y=-0.281 x+8.279, R2=0.9839.
2.4.2 astragalus polyose hydrochloric acid hydrolysis technology stability research
If table 5 shows, from the point of view of the change of molecular weight of astragalus polyose hydrolysate and the relative amount of each molecular weight section,
RSD value is smaller, wherein peak 2 be it is desirable that product, molecular weight ranges variation is all relatively large, but still at me
It is expected in the range of, the variation of the relative amount at peak 2 is smaller, illustrates that the technology stability is preferable, can satisfy astragalus polyose water
Solve the demand of product preparation.
5 astragalus polyose hydrochloric acid of table hydrolyzes technology stability research
2.4.3 astragalus polyose trifluoroacetic acid hydrolysis process stabilizing Journal of Sex Research
It is similar to Radix Astragali hydrochloric acid hydrolysis process results if table 6 shows, the RSD value of the relative amount at peak 2 and the change of molecular weight compared with
Small, average molecular weight is located at 3000 dalton or so, and relative amount accounts for about 60% or more in gross product, illustrates that the technique is steady
It is qualitative preferable, it can satisfy the demand of astragalus polyose hydrolysate preparation.
6 astragalus polyose trifluoroacetic acid hydrolysis process stabilizing Journal of Sex Research of table
2.4.3 conclusion
The above repeated experiment confirms the hydrochloric acid hydrolysis process of astragalus polyose, and trifluoroacetic acid hydrolysis technique has preferable weight
Renaturation is suitble to industrial production and the preparation of middle-molecular-weihydroxyethyl section astragalus polyose.
The experimental study of influence of the acid hydrolysis products of three astragalus polyoses to immunosuppressed mice mucosal immunity function
3.1 experimental materials: the preparation of astragalus polyose is the same as 1.1 methods.Astragalus polyose acid hydrolysate prepares same 2.3.1, Huang
Astragalus polysaccharides acid hydrolysate is respectively 1KD with molecular cut off and the bag filter dialysis treatment of 10KD, and the molecular weight for obtaining purifying is situated between
In 103~104Middle-molecular-weihydroxyethyl section astragalus polyose between dalton, through detecting, middle-molecular-weihydroxyethyl is between 103~104Polysaccharide phase
To content 90%.
Major experimental reagent: Tris Base (Sigma), glycine (sigma), albumen Marker (Thermo), ZO-
1Antibody (Affinity Biosciences), anti-beta-actin rabbit polyclonal antibody (Affinity Biosciences),
HRP Goat Anti-Rabbit IgG Antibody (Abcam), pvdf membrane (Millipore), ethyl alcohol (Tianjin Ke Miou),
Dimethylbenzene (Tianjin Ke Miou), ether (Tianjin richness space chemical industry), hematoxylin (Beijing Suo Laibao), Yihong (Beijing Suo Laibao), BCA
Protein quantification kit (the green skies), SIgA Elisa kit (Shanghai enzyme is far biological), IFN-γ Elisa kit (Shanghai
Enzyme is far biological), IL-4Elisa kit (Shanghai enzyme is far biological), IL-2Elisa kit (enzyme is far biological), ET kit
(enzyme is far biological), gel reagent preparation box (Xi'an doctor's moral), RIPA proteolytic agent box (Xi'an doctor's moral), Tween-20
(Chinese medicines group), cyclophosphamide (Shanghai source leaf biology), faeces DNA extracts kit (Tiangeng biochemical technology), nucleic acid dye
Gene Green (Tiangeng biochemical technology), 2000bp DNA Marker (Tiangeng biochemical technology), Loading buffer (Tiangeng
Biochemical technology), acetic acid mark product (Tianjin Ke Miou), propionic acid mark product (Tianjin Ke Miou), butyric acid mark product (Tianjin Ke Miou).
3.2 experimental animal
Female KM mouse, 18g ± 2g are purchased from The Fourth Military Medical University's Experimental Animal Center, animal productiong licensing number
SYXK (Shan) 2014-001.
The grouping of 3.3 experimental animals and processing
Female KM mouse, weight 18g ± 2g adapt to raising after a week, carry out being layered grouping, number at random, every group 12
Only.It is divided into Normal group, model group, positive drug group, astragalus polyose (APS) high and low dose group, middle-molecular-weihydroxyethyl section astragalus polyose
(DAPS) high and low dose group, intraperitoneal injection of cyclophosphamide induce immunosuppressant animal model, groups of animals processing method such as table 8.
Mouse weight of every two days records.Successive administration 30 days, after drug therapy, sterile collection stool in mice.Then to small
Mouse is deprived of food but not water 12 hours, records the weight of every mouse, and mouse carries out eyeball and cervical dislocation after blood is taken to put to death, and separates serum,
It takes the thymus gland of mouse and spleen and weighs；Animal small intestine jejunal segment 20cm or so is intercepted, this section tissue is rinsed with PBS, returns
Receive intestines mucus；Jejunal segment tissue freezing is stand-by；About 2 cm of the complete jejunal segment of animal is intercepted, is soaked in 4% paraformaldehyde, it is fixed
Processing, for subsequent slice production observation.
The grouping of 8 experimental animal of table and dosage regimen
Note: N.control: Normal group；Model: model group；P.control: positive controls；APS(100):
Astragalus polyose low dose group；APS (400): astragalus polyose high dose group；DAPS (100): middle-molecular-weihydroxyethyl section astragalus polyose low dosage
Group；DAPS (400): middle-molecular-weihydroxyethyl section astragalus polyose high dose group
3.4 experiment test indexs and method
3.4.1 mouse intestinal mucosa morphology is observed
Take the mouse jejunum that fixes according to routine paraffin wax microsection manufacture method through dehydration, transparent, waxdip, embedding, slice,
Mounting after HE dyeing, microscopically observation, villus length (V), the Crypt depth (C) of measurement record small intestine.
3.4.2 Elisa method detection astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose to mouse intestines mucus SIgA, IFN- γ,
The influence of IL-4 content
Illustrate to operate according to corresponding kit, detect mouse casing slime SIgA, IFN-γ, IL-4 content respectively, utilizes
In BCA protein quantification kit measurement casing slime after protein content, SIgA, IFN-γ, IL-4 in unit of account protein.
3.4.3 detection-Western blot the method for mouse small intestine tissue tight connection albumen ZO-1
It extracts small intestine's albumen: the freezing of 100mg mouse intestinal tissue being ground to fine-powdered with liquid nitrogen, 750 μ L are added
(750 μ L lysates, 7.5 μ L PMSF (phenylmethylsulfonyl fluoride, protease inhibitors) are using in first several minutes for RIPA lysate
It is added.Continue to be ground to intestinal tissue and be in a liquid state, suck in EP pipe, 4 DEG C of centrifuge 14000r/min, is centrifuged 5min.Draw supernatant
Liquid therefrom takes 10 μ L for protein quantification, and remaining supernatant is distributed into multiple EP pipes as unit of 30 μ L, and -80 DEG C of refrigerators freeze
It deposits stand-by.
The intestinal tissue albumen of extraction is after polyacrylamide gel electrophoresis separates, through transferring film, closing, primary antibody reaction, secondary antibody
It takes pictures after reaction, luminescence-producing reaction, development, fixing, gel images processing system analyzes the molecular weight and net OD value of object tape.
3.4.4 the influence of astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose to mouse intestinal flora -- 16SrRNA Miseq
Illumina PCR sequencing PCR
Stool in mice genomic DNA is extracted using faeces DNA extracts kit, with bacterium 16sDNAV3-V4 regional sequence
Design primer, primer sequence are as follows: upstream: ACTCCTACGGGAGGCAGCA, downstream: GGACTACHVGGGTWTCTAAT.Into
Row PCR amplification.Amplification condition are as follows: 95 DEG C, 3min (denaturation)；95 DEG C, 30s (denaturation) -- 55 DEG C, 30 s (annealing) --- 72 DEG C,
45s (extension)；After 30 circulations, 72 DEG C, 10min.PCR amplification system and amplification condition are as follows:
9 PCR amplification system of table composition
After the completion of amplification, PCR product is purified, is quantified, uniforming analysis；Then Miseq illimina survey is carried out
Sequence (being completed by Shanghai Major Biological Medical Technology Co., Ltd.) simultaneously carries out enterobacteriaceae identification and relevant information credit analysis, detects intestines
The variation of road bacterial diversity and structure of community.
3.4.5 in stool in mice SCFA (short chain fatty acids) measurement
200mg stool in mice is substantially soluble in 1.5mL chromatographic ethanol, is centrifuged (10000r/min, 5 min), in 4 DEG C of rings
30min is stood under border, with 1mL syringe Aspirate supernatant, is filtered with 0.22 μm of filter spare into gas phase bottle.Chromatostrip
Part: chromatographic column AT-WaX (30m × 0.25mm × 0.25 μm)；Chromatographic column temperature program: 40 DEG C of initial temperature, 1min is kept；With
The rate of 8 DEG C/min rises to 180 DEG C, keeps 1min；It is heated up 200 DEG C with the rate of 20 DEG C/min, keeps 5min.Carrier gas: He；
Flow rate of carrier gas: 1.2mL/min；Injector temperature: 200 DEG C；Input mode: without shunting；Sample volume: 1 μ L；Detector temperature
(FID): 230 DEG C.Quantitative analysis after using acetic acid, propionic acid, butyric acid as standard items sample introduction.
3.5 experimental result
3.5.1 the influence of astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose to mouse intestinal mucosa morphology
Animal (N.control) intestinal villus grown under natural conditions is close, neat, complete；The mouse of CY processing
(Model) intestinal villus shorten, overstriking, swelling, atrophy, fracture；By LIVZON CHANGLE (P.control), astragalus polyose, middle molecule
After measuring section Effect of APS in Treating, intestinal villi length has obvious recovery trend, and swelling, atrophy, phenomenon of rupture also subtract in various degree
Light even to disappear, villus arrangement architecture also restores neat.
Astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose to mouse small intestine villus length (V), Crypt depth (C), V/C shadow
It rings as shown in table 10.The results show that the V value of model group reduces, difference has significant meaning compared with normal group.Each drug-treated group
V value can be improved, data difference has significant meaning.Model group C increases compared with normal group, and each drug-treated group C value is compared with mould
Type group decreases, and in addition to astragalus polyose (100 mg/kg) group, difference has significant meaning.Compared with normal group, model group
The decline of V/C value, V/C value can be improved in the processing of each medicine group, and data difference has significant meaning.
Influence of table 10 APS and DAPS to immunosuppressed mice small intestine V, C, V/C
N.control: Normal group；Model: model group；P.control: positive controls；DAPS (mg/kg): in
Molecular weight section astragalus polyose group；APS (mg/kg): astragalus polyose group
Note: with normal group ratio, #p < 0.05, ##p < 0.01；With model group ratio, * p < 0.05, * * p < 0.01. Note:
Compared with normal control, #p < 0.05, ##p < 0.01；Compared with model control,*
P < 0.05, * * p < 0.01.
3.5.2 the influence of astragalus polyose, DAPS to mouse intestines mucus SIgA, IFN-γ, IL-4 content
As shown in table 11, SIgA secretory volume is remarkably decreased in model group casing slime, and the SIgA secretory volume of remaining each group is higher than
Model group, data difference have significant meaning, and compared with APS (400), SIgA content is higher in DAPS (400) intestines mucus, poor
It is different to have significant meaning.In Fig. 2, with normal group ratio, #p < 0.05；With model group ratio, * * p < 0.01；With astragalus polyose (400) group
Than △ p < 0.05.
Influence of table 11 APS and DAPS to SIgA content in immunosuppressed mice casing slime
Such as
Shown in table 12, IFN-γ/IL-4 value of model group mouse is remarkably decreased, and IFN-γ/IL-4 of remaining each group is equal
Restored, data difference has significant meaning, compared with APS (400), in DAPS (400) intestines mucus IFN-γ/IL-4 compared with
Height, difference have significant meaning.
Table 12 APS and DAPS is to IFN-γ/IL-4 influence in immunosuppressed mice casing slime
As shown in Figure 1, immunosuppressed mice intestinal mucosal mechanical barrier is impaired, small intestine's tight junction protein ZO-1 table
Obvious up to decline, after drug-treated, the expression of tight junction protein is restored, and wherein middle-molecular-weihydroxyethyl section astragalus polyose is extensive
Multiple effect is better than APS.In Fig. 1, N.control: Normal group；Model: model group；P.control: positive controls；
DAPS: middle-molecular-weihydroxyethyl section astragalus polyose group；APS: astragalus polyose group
Note: with normal group ratio, ##p < 0.01；With model group ratio, * p < 0.05, * * p < 0.01；With APS (400) group
Than △ p < 0.05；Compared with APS (100), ▽ p < 0.05.N=3).
3.5.4 the influence of astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose to mouse intestinal flora
Sample dilution curve data analysis when to mouse intestinal flora 16SrRNA sequencing is learnt, with sequencing sequence number
The raising of amount, OUT number of levels no longer increase, and dilution curve tends to flat, indicate that sequencing data is reasonable, subsequent data analysis
It is meaningful.The description multifarious index of intestinal microflora has multiple, and wherein Ace, Chao index represent the rich of group
Fu Du, Shannon, Simpson index represent the diversity of group.Ace, Chao index are bigger, and it is higher to represent richness；
Shannon index is bigger, indicates that community diversity is higher；Simpson index is smaller, and the diversity for representing group is higher.From Fig. 2
It follows that the richness (Ace, Chao) of Model has decreasing trend (p < 0.05) compared with Ncontrol, and
Pcontrol, middle-molecular-weihydroxyethyl section astragalus polyose, astragalus polyose chao index conspicuousness increase (p < 0.05)； Pcontrol,
The Ace index conspicuousness of middle-molecular-weihydroxyethyl section astragalus polyose increases (p < 0.05)；The Ace index of astragalus polyose has recovery trend,
But there was no significant difference.Community diversity index Shannon index variation difference is smaller between each group, and Simpson index can be seen
Out, the diversity of model group has downward trend, and only middle-molecular-weihydroxyethyl section astragalus polyose group, which is shown, has certain recovery to make Simpson
With.
PCoA analysis (principal co-ordinates analysis) is used to study the phase of each group intestinal flora composition
Like property and otherness.Relative distance in PCoA figure between horizontal, axis of ordinates each sample of number, distance is closer, indicates two groups
Similitude is higher, on the contrary then illustrate that otherness is larger.The percentage of transverse and longitudinal coordinate, which represents, causes otherness between different groups
Different factor proportions.Fig. 3 is the enterobacteriaceae structure PCoA analysis chart between each group, as seen from the figure, Model with
Ncontrol distance farther out, Pcontrol, middle-molecular-weihydroxyethyl section astragalus polyose and Ncontrol distance relatively, recovery trend compared with
Obviously, and astragalus polyose then to restore trend weaker.
All groups of areas sample faeces DNA V3-V4 are detected by Miseq illumina sequencing, detect 8 altogether
Bacterium door less than 1% is attributed to others by main bacterium door.This eight bacterium doors are Deferribacteres (deferrization bar respectively
Bacterium door), Spirochaetae (spirillum door), Fusobacteria (Fusobacterium door), Proteobacteria (Proteobacteria),
Actinobacteria (actinomyces door), Firmicutes (Firmicutes), Bacteroidetes (Bacteroidetes),
unclassified_k__norank.The variation of Firmicutes/ Bacteroidetes in each group is counted, such as table 13
Show.Compared with Ncontrol, the Firmicutes/Bacteroidetes ratio in Model is in extremely significant increase (p <
0.01)；Compared with Model, under Firmicutes/Bacteroidetes ratio is significant or extremely significant in Pcontrol, DAPS
It drops (p < 0.05, p < 0.01), has apparent recovery trend, the APS ratio does not decline；Compared with APS, under DAPS conspicuousness
It drops (p < 0.05).
Influence of table 13 APS and DAPS to Firmicutes/Bacteroidetes
APS: astragalus polyose group；DAPS: middle-molecular-weihydroxyethyl section astragalus polyose group；Ncontrol: Normal group； Model:
Model group；Pcontrol: positive controls
Note: with normal group ratio, ##p < 0.01；With model group ratio, * p < 0.05, * * p < 0.01；With APS ratio, △ p <
0.05。
Belong to level on analysis shows that, the Pseudomonas to relative amount less than 1% is included into others, the total inspection greater than 1%
Measure 35 kinds of Pseudomonas.Table 14 is counted to the significant Pseudomonas of content is changed in each group.Compared with Ncontrol, Model
What middle relative amount changed greatly mainly has: Bacteroides, Lactobacillus, prevotellaceae_UGG-001,
norank_f_Bacteroidales_S24-7_group、Odoribacter、 Lachnospiraceae_NK4A136_
The relative abundance of group, Alloprevolella etc. decline (p < 0.05 or p < 0.01), and Rhodococcus relative abundance increases
Add (p < 0.05).Compared with Model, the variation of Pcontrol, DAPS Pseudomonas has apparent recovery trend (p < 0.05 or p <
0.01), Lactobacillus recovery effects are obvious (p < 0.05) in APS group, not to the recovery trend of other a few class Pseudomonas
Obviously.Compared with APS, Bacteroides, prevotellaceae_UGG-001, norank_f_ in DAPS
Bacteroidales_S24-7_group、Odoribacter、Lachnospiraceae_NK4A136_group、
Alloprevolella is significant or extremely significant increase (p < 0.05 or p < 0.01), Rhodococcus conspicuousness decline (p <
0.05).In conclusion DAPS belongs to horizontal disorder with apparent restitution to intestinal flora, effect is better than APS.
Influence of table 14 APS and DAPS to level is belonged to
APS: astragalus polyose group；DAPS: middle-molecular-weihydroxyethyl section astragalus polyose group；Ncontrol: Normal group； Model:
Model group；Pcontrol: positive controls；With normal group ratio, #p < 0.05, ##p < 0.01；With model group ratio, * p <
0.05, * * p < 0.01；With APS ratio, △ p < 0.05, △ △ p < 0.01.
3.5.5 in stool in mice SCFA (short chain fatty acids) measurement
Sample introduction is distinguished using standard acetic acid, propionic acid and butyric acid, and standard curve is respectively as follows: y=0.00000418 x+
0.52159094, R2=0.999 (acetic acid)；Y=0.00000013x+0.02089694, R2=0.999 (propionic acid)；Y=
0.00000011x+0.01671176, R2=0.999 (butyric acid).
Short chain fatty acids (SCFA) are intestinal floras using carbohydrate as the tunning of substrate, mainly comprising acetic acid,
Propionic acid, butyric acid, valeric acid etc..Relative amount of the SCFA in each group is acquired according to mark song.As shown in figure 4, with N.control phase
Than acetic acid (acetic acid), propionic acid (propionic acid), butyric acid in the immunosuppressed mice excrement of CY induction
(butyrate) content is in the trend that is decreased obviously (p < 0.05).APS in Fig. 4: astragalus polyose group；DAPS: middle-molecular-weihydroxyethyl section is yellow
Astragalus polysaccharides group；N.control: Normal group；Model: model group；P.control: positive controls；
Note: with normal group ratio, #p < 0.05, ##p < 0.01；With model group ratio, * p < 0.05, * * p < 0.01；With APS
(400) compare, △ p < 0.05.
After astragalus polyose and the processing of middle-molecular-weihydroxyethyl section astragalus polyose, excrement Short-Chain Fatty Acids content is risen, with
Model compares, and data difference has significant meaning (p < 0.05).Therefore astragalus polyose, middle-molecular-weihydroxyethyl section astragalus polyose can be extensive
The multiple immunosuppressed mice intestinal tract flora metabolite SCFA as caused by CY is reduced, the results show that middle-molecular-weihydroxyethyl section astragalus polyose
Recovery effects be better than astragalus polyose.
3.6 conclusion
Astragalus polyose has Immune enhancement function；Astragalus polyose is disorderly to the intestinal flora for the immune mouse processed that cyclophosphamide induces
It disorderly also has a certain impact, the generation of enterobacteriaceae metabolin short chain fatty acids can be promoted；Therefore, astragalus polyose is to mucosal immunity
Influence of the influence of function to astragalus polyose to intestinal flora Tiny ecosystem is related；In vitro intestines culture experiment confirms that astragalus polyose can
To promote the secretion of SIgA, enhance Gut mucosal immune function.In many index detected, if SIgA secretes, IFN-γ/
IL-4, the expression of small intestine's tight junction protein ZO-1, many indexs such as intestinal microecology variation, SCFA content index and spleen index
In, show that middle-molecular-weihydroxyethyl section astragalus polyose, better than astragalus polyose, illustrates astragalus polyose to the recovery Effects of immunosuppressed mice
Hydrolysate has the effect for preferably promoting mucosa-immune function.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (8)
1. a kind of middle-molecular-weihydroxyethyl section astragalus polyose, which is characterized in that it is hydrolyzed and is obtained by Radix Astragali Thick many candies, the middle-molecular-weihydroxyethyl section Radix Astragali
The molecular weight ranges of polysaccharide are 103-104Dalton.
2. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose, which is characterized in that first by Radix Astragali Thick many candies hot water dissolving, acid adding
And heating water bath, lye adjusting pH is then added and pulverizes and sieves after hydrolyzate concentrate drying to neutrality, obtains astragalus polyose water
Object is solved, the content of middle-molecular-weihydroxyethyl section astragalus polyose is not less than 50% in the astragalus polyose hydrolysate.
3. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose as claimed in claim 2, which is characterized in that the hydrolyzate
Dialysis treatment has also been carried out before drying after concentration, has obtained middle-molecular-weihydroxyethyl section astragalus polyose after purification.
4. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose as claimed in claim 2, which is characterized in that the acid is energy
The organic acid and inorganic acid of hydrolyzing glucosidic bonds.
5. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose as claimed in claim 2, which is characterized in that the acid is salt
Acid, being adjusted to concentration of hydrochloric acid in Radix Astragali Thick many candies solution is 0.03~0.30mol/L, and water bath heating temperature is 10~100 DEG C, the time
For 1~8h.
6. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose as claimed in claim 2, which is characterized in that the acid is three
Fluoroacetic acid, being adjusted to trifluoroacetic acid concentration in Radix Astragali Thick many candies solution is 0.05~0.30mol/L, water bath heating temperature is 10~
100 DEG C, the time is 1~8h.
7. a kind of preparation method of middle-molecular-weihydroxyethyl section astragalus polyose as claimed in claim 2, which is characterized in that the Radix Astragali is thick
The extracting method of polysaccharide is as follows: water temperature extraction takes after Milkvetch Root is crushed, concentrated extracting solution, removing protein, centrifugation, and supernatant is dense
After contracting, is precipitated through 80% ethanol washing, obtain Radix Astragali Thick many candies.
8. middle-molecular-weihydroxyethyl section astragalus polyose as described in claim 1 is in preparation for improving Mucosal Immunity and intestinal microecology
The drug of system function or the application in food.
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| CN116217746A (en) * | 2023-02-07 | 2023-06-06 | 赤峰学院 | Fungus polysaccharide refining and purifying process |
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