CN109265576A - A kind of macromolecule astragalus polyose and the preparation method and application thereof with anti-inflammatory activity - Google Patents
A kind of macromolecule astragalus polyose and the preparation method and application thereof with anti-inflammatory activity Download PDFInfo
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- CN109265576A CN109265576A CN201811130205.6A CN201811130205A CN109265576A CN 109265576 A CN109265576 A CN 109265576A CN 201811130205 A CN201811130205 A CN 201811130205A CN 109265576 A CN109265576 A CN 109265576A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The present invention discloses a kind of macromolecule astragalus polyose and the preparation method and application thereof with anti-inflammatory activity, belongs to natural drug and isolates and purifies field.The molecular weight of the macromolecule astragalus polyose is 2,000~4,000kDa, and purity is 75% or more.The present invention also provides the preparation methods and application of a kind of macromolecule astragalus polyose, this method obtains the APS of macromolecule with water extraction and alcohol precipitation method extraction purification, the macromolecule astragalus polyose of high-purity can be obtained in the method for the DEAE-52 chromatographic column combination HW-65F chromatography column separating purification used, and stablize feasible, products obtained therefrom purity is higher, furthermore it and by high-purity Huang Qi Jing Polysaccharide A PS 3a acts in the mouse colitis model by DSS induction, it is found to have preferable anti-inflammatory activity, can be used for preparing anti-inflammatory drug.
Description
Technical field
The invention belongs to natural drugs to isolate and purify field, and in particular to a kind of macromolecule Radix Astragali with anti-inflammatory activity
Polysaccharide and the preparation method and application thereof.
Background technique
Radix Astragali comes from leguminous mongolian scutellaria (Astragalus membranaceus (Fisch.)
Bge.var.mongholicus (Bge.) Hsiao) or Astragalus membranacus (Astragalus membranaceus (Fisch.)
Bge. dry root), sweet in flavor first recorded in Shennong's Herbal, tepor, returns spleen, lung channel, have invigorate the spleen and benefit qi, inducing diuresis for removing edema, support
Taking poison and growing muscle due and other effects has used more than 2,000 years so far.Flavonoids, saponins, polysaccharide and some micro are mainly contained in Radix Astragali
Element etc..And primary water-soluble ingredient of the astragalus polyose (Astragalus polysaccharides, APS) as Radix Astragali,
It plays a significant role in many activity.
APS polarity is bigger than normal, relatively water extraction is mostly used to extract, can after direct alcohol precipitation or classification alcohol precipitation after Aqueous extracts concentration
Obtain the APS of molecular weight difference section.Astragalus polyose carries out de- albumen processing after water extract-alcohol precipitation, as enzyme process, Sevage method,
Trichloroacetic acid method is all the method that removing protein is often selected, but every kind of single method has a certain limitation, enzyme process with
Sevage method is combined the effect that polysaccharide purification can be improved.Astragalus polyose after removing protein dialysed, be freeze-dried after up to Radix Astragali
Thick many candies.Radix Astragali Thick many candies are successively through the higher Huang Qi Jing polysaccharide of the available purity of chromatography column packing of different nature, usually
Wire feeding has ion-exchange chromatography, the most frequently used with DEAE- cellulose, and gel permeation chromatography filling kind is relatively abundant, often
With having glucose gel (Sephadex G), polyacrylamide glucose gel (Sephacryl HR) and Ago-Gel
(Sepharose), but the separating ranges of these fillers generally arrive hundreds of thousands dalton (Da) thousands of, and it is more not to be suitable for macromolecular
The separation of sugar.Since the structure of molecular weight upper section (molecular weight is in million grades of Da) is more complicated, isolate and purify it is more difficult,
Corresponding research is very few, and the research of APS at present is the lower part of molecular weight (molecular weight is differed thousands of to hundreds of thousands Da) mostly,
And since the structure of molecular weight upper section (molecular weight is at million grades) is more complicated, isolate and purify more difficult, corresponding research
Very few, therefore, it is that there are certain challenges for a big difficulty that the purification of the APS of high molecular weight is probed into preparation and its activity.
Previous report focuses mostly in terms of the activity of the astragalus polyose of small-molecular-weight, such as anti-oxidant, anti-inflammatory, antitumor
Aspect, and the polysaccharide anti-inflammatory activity of its macromolecule there is no report.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of with anti-inflammatory activity
Macromolecule astragalus polyose.
Another object of the present invention is to provide the preparation methods of the above-mentioned macromolecule astragalus polyose with anti-inflammatory activity.
A further object of the present invention is to provide the applications of the above-mentioned macromolecule astragalus polyose with anti-inflammatory activity.
The present invention provides the extraction purification technology of macromolecule (2,000~4,000kDa) APS a kind of, and this method stabilization can
Row, products obtained therefrom purity is higher, furthermore and by high-purity macromolecule APS acts in the colitis model of DSS inducing mouse and sends out
Now there is preferable anti-inflammatory activity.
The purpose of the invention is achieved by the following technical solution:
A kind of macromolecule astragalus polyose with anti-inflammatory activity, molecular weight 2,000~4,000kDa, purity are
75% or more.
Preferably, molecular weight 2,700~3,700kDa, purity are 75% or more.
It is furthermore preferred that its molecular weight is 3,300~3,500kDa, purity are 85% or more.
Further, molecular weight 3,300~3,500kDa, purity are 88% or more.
Most preferably, molecular weight 3,388kDa, purity are 88% or more.
A kind of preparation method of the macromolecule astragalus polyose with anti-inflammatory activity, includes the following steps:
By Radix Astragali Thick many candies on DEAE-52 chromatographic column with 0.05M sodium chloride, 0.15M sodium chloride, 0.3M sodium chloride successively
3 Polysaccharide A SP 1, APS 2, APS 3 are respectively obtained after elution, are freezed after desalination wherein the highest APS 3 of yield is dialysed
It is dry；Wherein APS 3 is eluted with water through HW-65F chromatographic column, obtains Huang Qi Jing Polysaccharide A PS 3a.
Preferably, DEAE-52 chromatographic column (2.5 × 40cm) flow velocity is 1~2mL/min, HW-65F chromatographic column
(1.5 × 100cm) flow velocity is 0.4~0.6mL/min.
Preferably, the time of the dialysis is 24~48h；More preferably 48h.
Preferably, the APS 3 is the solution for being 10~30mg/mL by 3 compound concentration of APS through HW-65F chromatographic column
By HW-65F chromatographic column.
The preparation method of the Radix Astragali Thick many candies, includes the following steps:
Radix Astragali dry root is carried out refluxing extraction 3~5 times with 70% ethyl alcohol of 8~10 times of medicinal material amounts at 75~85 DEG C,
1~3h every time；After alcohol extracting, medicinal material is dried, water is carried out with same method and mentions, dehydrated alcohol, which is added, after Aqueous extracts are concentrated makes its concentration
For 70% and in low temperature alcohol precipitation stay overnight, next day centrifugation, precipitating is redissolved with water, a certain amount of papain enzymolysis is added, then
De- albumen is carried out with Sevage method；Polysaccharide solution after de- albumen is freeze-dried after flowing water is dialysed to get Radix Astragali Thick many candies.
Preferably, the temperature of the reflux is 80 DEG C.
Preferably, the temperature of the low temperature is 2~8 DEG C；More preferably 4 DEG C.
Preferably, the dosage of the papain is 50~60U/g Radix Astragali.
Preferably, the condition of the enzymatic hydrolysis is that 4~6h is digested at 60 DEG C；More preferably 5h is digested at 60 DEG C.
Preferably, the Sevage method is by n-butanol-chloroform (4:1, v/v) mixed liquor of 1/5 amount of addition in solution
And shaking out；
Preferably, the time through flowing water dialysis is 24~48h；More preferably 48h.
The macromolecule astragalus polyose application in preparing anti-inflammatory drugs with anti-inflammatory activity.
Huang Qi Jing Polysaccharide A PS 3a is acted in the mouse colitis model by DSS induction, is measured with ELISA kit small
TNF-α expression quantity in mouse colonic tissue, APS 3a can significantly reduce TNF-α expression quantity as the result is shown, show there is certain anti-inflammatory work
Property.
The present invention has the following advantages and effects with respect to the prior art:
The present invention obtains the APS of macromolecule with water extraction and alcohol precipitation method extraction purification, and the DEAE-52 chromatographic column used combines
HW-65F chromatographs the macromolecule astragalus polyose of the available high-purity of method of column separating purification, and this polysaccharide is tentatively living
Property evaluation in be found to have good anti-inflammatory effect.
Detailed description of the invention
Fig. 1 is the extraction process route map of Huang Qi Jing Polysaccharide A PS 3a.
Fig. 2 is DEAE-52 chromatographic column elution curve, is followed successively by APS 1, APS 2, APS 3 from left to right.
Fig. 3 is APS 3a gel liquid chromatogram.
Fig. 4 is APS 3a1H-NMR spectrum.
Fig. 5 is TNF-α expression quantity in mouse Colon tissue (P < 0.05 *, compared with model group).
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by factory.Used material, reagent etc. are unless otherwise instructed the examination obtained from commercial channels
Agent and material.
The extraction process route map of Huang Qi Jing Polysaccharide A PS 3a of the invention is as shown in Figure 1.
Embodiment 1
1,50g Radix Astragali dry root carries out refluxing extraction 3 times with 70% ethyl alcohol of 400mL at 80 DEG C, each 1h.After alcohol extracting,
Medicinal material is dried, water is carried out with same method and mentions, Aqueous extracts are concentrated to make its concentration 70% to dehydrated alcohol is added after 50~60mL
And alcohol precipitation is stayed overnight in 4 DEG C of refrigerators, next day centrifugation, centrifuged deposit volatilizes ethyl alcohol and is redissolved with water, and 2500~3000U pawpaw is added
Protease digests 5h at 60 DEG C, then (is mixed n-butanol-chloroform (4:1, v/v) that 1/5 amount is added in solution with Sevage method
Liquid and shaking out) it carries out de- albumen 3 times.Polysaccharide solution after de- albumen is freeze-dried after flowing water dialyses 48h to get Radix Astragali
Thick many candies, purity 39.68%.
2,0.1g Radix Astragali Thick many candies on DEAE-52 anion exchange chromatography with 0.05M sodium chloride, 0.15M sodium chloride,
0.3M sodium chloride successively elutes, and every 5min 1 is managed, every pipe 7mL, and each gradient detects polysaccharide with phend-sulphuric acid respectively, respectively
To 3 sections of Polysaccharide A SP 1, APS 2, APS 3 (Fig. 2), will be freeze-dried after the dialysis 48h desalination of APS 3.After freeze-drying
APS 3 detects polysaccharide with phend-sulphuric acid, and obtaining purity is about 77%.The solution 1mL for being 10mg/mL by 3 compound concentration of APS
And through TOYOPEARL HW-65F exclusion chromatography column, it is eluted with water, every 15min mono- is managed, and APS 3a molecule can be obtained in every pipe 6mL
Amount is 3,388kDa, purity 88%.
Wherein, DEAE-52 chromatographic column (2.5 × 40cm) flow velocity is 1~2mL/min, TOYOPEARL HW-65F chromatographic column
(1.5 × 100cm) flow velocity is 0.4~0.6mL/min.
APS 3a gel liquid chromatogram is as shown in Figure 3, the results showed that: for single symmetrical peak, show that purity is higher.
APS 3a 1H-NMR spectrum is as shown in Figure 4, the results showed that: chemical shift δ 4.46ppm, δ 5.14ppm, δ in figure
5.16ppm, δ 5.2ppm, δ 5.23ppm proton signal correspond respectively to galactopyranose, glucopyranose, α-grape
The H-1 signal of sugar, α-xylose, β-arabinose and furan type arabinose.
Embodiment 2
500g Radix Astragali dry root carries out refluxing extraction 6h with 70% ethyl alcohol of 4L at 80 DEG C.After alcohol extracting, medicinal material is dried,
Water being carried out with same method to mention, addition dehydrated alcohol makes its concentration 70% after Aqueous extracts are concentrated and alcohol precipitation is stayed overnight in 4 DEG C of refrigerators,
Next day centrifugation, centrifuged deposit volatilize ethyl alcohol and are redissolved with water, and 25000~30000U papain is added and digests at 60 DEG C
5h, then carried out with Sevage method (n-butanol-chloroform (4:1, v/v) mixed liquor and the shaking out of 1/5 amount will be added in solution)
De- albumen 3 times.To get Radix Astragali Thick many candies, purity is polysaccharide solution after de- albumen for freeze-drying after flowing water dialyses 48h
41%.
Embodiment 3
Radix Astragali Thick many candies made from 0.5g embodiment 1 on DEAE-52 anion exchange chromatography with 0.05M sodium chloride,
0.15M sodium chloride, 0.3M sodium chloride successively elute, and every 3min 1 is managed, and every pipe 7mL, each gradient is detected with phend-sulphuric acid respectively
Polysaccharide respectively obtains 3 sections of Polysaccharide A SP 1, APS 2, APS 3, and APS 3 is dialysed and is freeze-dried after 48h desalination.
Embodiment 4
It is 20~30mg/mL by 3 compound concentration of APS after being freeze-dried in embodiment 1,3 solution of 1mL APS is through HW-
65F exclusion chromatography column, is eluted with water, and every 15min mono- is managed, every pipe 6mL, obtains Huang Qi Jing Polysaccharide A PS 3a, molecular weight 2,
187kDa, purity 89%.
Embodiment 5
APS 3a in embodiment 1 is applied to first to evaluate by the mouse colitis model that DSS (dextran sulfate sodium) induces
Step activity.20 C57BL/6 female mices are randomly divided into four groups, every group 5, respectively blank group (Control group),
3%DSS model group (Model group), 3%DSS+5mg/kg Dexamethasone group (Dexa-treatment group), 3%
DSS+200mg/kg APS 3a group (APS 3a-treatment group).Totally one week experimental period, mouse is taken after experiment
Colonic tissue, tissue homogenate are used for cytokines measurement.With TNF-α expression in ELISA kit measurement mouse Colon tissue
Amount shows there is certain anti-inflammatory activity as a result as shown in figure 5, display APS 3a can significantly reduce TNF-α expression quantity.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of macromolecule astragalus polyose with anti-inflammatory activity, it is characterised in that point of the macromolecule astragalus polyose
Son amount is 2,000~4,000kDa, and purity is 75% or more.
2. the macromolecule astragalus polyose according to claim 1 with anti-inflammatory activity, it is characterised in that:
The molecular weight of the macromolecule astragalus polyose is 2,700~3,700kDa, and purity is 75% or more.
3. the macromolecule astragalus polyose according to claim 1 with anti-inflammatory activity, it is characterised in that:
The molecular weight of the macromolecule astragalus polyose is 3,300~3,500kDa, and purity is 85% or more.
4. a kind of preparation method of the macromolecule astragalus polyose with anti-inflammatory activity, it is characterised in that include the following steps:
Radix Astragali Thick many candies are successively eluted on DEAE-52 chromatographic column with 0.05M sodium chloride, 0.15M sodium chloride, 0.3M sodium chloride
After respectively obtain 3 Polysaccharide A SP 1, APS 2, APS 3, be freeze-dried after desalination wherein the highest APS 3 of yield is dialysed；
Wherein APS 3 is eluted with water through HW-65F chromatographic column, obtains Huang Qi Jing Polysaccharide A PS 3a.
5. the preparation method according to claim 4, it is characterised in that:
The DEAE-52 chromatography column flow rate is 1~2mL/min, and it is 0.4~0.6mL/min that HW-65F, which chromatographs column flow rate,；
The time of the dialysis is 24~48h；
The APS 3 through HW-65F chromatographic column be by 3 compound concentration of APS be 10~30mg/mL solution after through HW-65F layers
Analyse column.
6. preparation method according to claim 4 or 5, it is characterised in that:
The preparation method of the Radix Astragali Thick many candies, includes the following steps:
Radix Astragali dry root is carried out refluxing extraction 3~5 times with 70% ethyl alcohol of 8~10 times of medicinal material amounts at 75~85 DEG C, every time 1
~3h；After alcohol extracting, medicinal material is dried, water is carried out with same method and mentions, dehydrated alcohol, which is added, after Aqueous extracts are concentrated makes its concentration
70% and in low temperature alcohol precipitation stay overnight, next day centrifugation, precipitating is redissolved with water, a certain amount of papain enzymolysis is added, then use
Sevage method carries out de- albumen；Polysaccharide solution after de- albumen is freeze-dried after flowing water is dialysed to get Radix Astragali Thick many candies.
7. preparation method according to claim 6, it is characterised in that:
The temperature of the reflux is 80 DEG C；
The temperature of the low temperature is 2~8 DEG C.
8. preparation method according to claim 6, it is characterised in that:
The dosage of the papain is 50~60U/g Radix Astragali；
The condition of the enzymatic hydrolysis is that 4~6h is digested at 60 DEG C.
9. preparation method according to claim 6, it is characterised in that:
The Sevage method is by n-butanol-chloroform mixed liquor of the volume ratio 4:1 of 1/5 amount of addition in solution and to shake extraction
It takes；
The time through flowing water dialysis is 24~48h.
10. the described in any item macromolecule astragalus polyoses with anti-inflammatory activity of claims 1 to 3 are in preparing anti-inflammatory drug
Application.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113603802A (en) * | 2021-08-24 | 2021-11-05 | 华侨大学 | Preparation method and application of verbena polysaccharide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113603802A (en) * | 2021-08-24 | 2021-11-05 | 华侨大学 | Preparation method and application of verbena polysaccharide |
| CN113603802B (en) * | 2021-08-24 | 2022-10-14 | 华侨大学 | Preparation method and application of verbena polysaccharide |
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Application publication date: 20190125 |
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| RJ01 | Rejection of invention patent application after publication |