CN108892739A - A kind of method for extraction and purification of anemaran - Google Patents
A kind of method for extraction and purification of anemaran Download PDFInfo
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Abstract
The present invention provides a kind of method for extraction and purification of anemaran, including are extracted using organic solvent progress degreasing, water extraction, alcohol precipitating and obtained thick anemaran Common Anemarrhena Rhizome；And low molecular weight impurities are gone by dialysis membrane dialysis, DEAE- agarose Gel column adsorbing separation obtains anemaran desorbed solution, and dialysis membrane, which is dialysed, is made anemaran purification component.Present invention firstly provides the extracting methods of anemaran purification component, the anemaran obtained by this method refines component and shows compared with conventional method obtains thick anemaran to the higher activation capability of mouse macrophage RAW264.7, and to the stronger activation effect of expression for promoting iNOS and relevant cell factor mRNA.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method for extraction and purification of anemaran.
Background technique
Polysaccharide is a kind of natural polymer, it is made of aldose or ketose, is passed through by 10 or more monosaccharide
The polymer that glycosidic bond is formed by connecting is one of the four big base substances for constituting life, plays an important role in vital movement.People
It is merely observed that as the constituent of cell and the importance of energy substance for the early stage research of carbohydrate, find had later
A little polysaccharide can be used as the single-minded identification signal of cell surface, plays a part of to transmit information, takes part in cell in life science
Various activities have diversified biological function, such as polysaccharide has antiviral, anti-aging, anti-inflammatory, anticomplement, resists and burst
The effects of ulcer, hypoglycemic, blood pressure lowering and antithrombotic, there are also immunological regulation and antitumor biological effectiveness, it, which can be activated, exempts from
Epidemic disease receptor improves the immune function of body.
Polysaccharide is widely present in middle medicinal herbs, such as in rhizoma anemarrhenae.Rhizoma anemarrhenae is Liliaceae herbaceos perennial rhizoma anemarrhenae
The dry rhizome of (Anemarrhena asphodeloides Bunge), first recorded in《Sheng Nong's herbal classic》, middle product are classified as, property is bitter
It is cold, have the effects that the sliding dry, relieving restlessness of quenching the thirst of nourishing yin and lessening fire, ease constipation, sharp stool and urine, is Chinese medicine conventional medicament, is clinically used for exogenous disease
The symptoms such as pyreticosis, hyperpyrexia polydipsia, lung-heat type cough, osteopyrexia and fever, Heat Diabetes and dry constipation of intestines.In rhizoma anemarrhenae rich in steroid saponin,
The ingredients such as double benzene pyrrones, flavonoids, lignanoids, polysaccharide, organic acid and microelement.Simultaneously research shows that rhizoma anemarrhenae has
There are anti-inflammatory, anti-oxidant, anti-radiation, antiviral and antitumor, platelet aggregation-against and immunoregulation effect, memory can be improved
With senile dementia symptom, lipid-loweringing, antiatherosclerosis, improvement osteoporosis symptoms, hypoglycemic and treatment diabetes and other effects.
The method that number of patent application CN201110120368.8 extracts anemaran by rhizoma anemarrhenae medicine materical crude slice, this patent application is
It is considered as to put and regarding abandoning as failure, the invention discloses a kind of methods for extracting anemaran by rhizoma anemarrhenae medicine materical crude slice.This method process
Including：Rhizoma anemarrhenae medicine materical crude slice ethyl alcohol, which extracts, removes small molecule, and residue drying is extracted with deionized water, and extracting solution merges concentration, uses ethyl alcohol
Precipitating filters, obtains supernatant filter cake two parts, Washing of Filter Cake obtains the medicinal extract of the N1 containing neutral polysaccharide；Supernatant ethanol precipitation,
It filters, washing filter cake obtains the medicinal extract of the N2 containing neutral polysaccharide；Water is extracted after proposing residue drying with lye, in extracting solution hydrochloric acid
With to neutrality, is precipitated after concentration, filter, obtain supernatant filter cake two parts, Washing of Filter Cake obtains the leaching of the A1 containing acidic polysaccharose
Cream；Supernatant ethanol precipitation filters, and washing filter cake obtains the medicinal extract of the A2 containing acidic polysaccharose.The advantage of the invention is that using knowing
Female residue mentions in water and uses alkali carries on basis, improves the recovery rate of anemaran, enrich the type of polysaccharide in rhizoma anemarrhenae
And improve the content of polysaccharide.
A kind of anemaran extract of number of patent application CN201110087706.2 and preparation method thereof and medical usage, this
Invention is related to a kind of anemaran extract and preparation method and medical usage.After preparation method includes raw material degreasing, using
Water extracts, alcohol precipitates to obtain Thick many candies, and Thick many candies slough albumen, pigment by dialysis, ion-exchange chromatography, remove small molecule
It studies point, the anemaran extract of higher degree is made.The content of anemaran is in extract of the present invention with glucose meter, institute
The content for stating polysaccharide accounts for 60% or more anemarrhena asphodefoides extract weight percent.The present invention is had found by a large amount of pharmacological experiment, is known
Female polyoses extract has immunosuppressive activity, for treating immunoinflammatory injury disease：Ephritis, pneumonia, upper respiratory tract sense
The medical usage of dye, hepatitis, tonsillitis and diabetes.
It is only to have carried out extracting research to anemaran that two above, which is applied for a patent, lacks purification step, is only capable of obtaining thick
Anemaran, while also lacking the method for measuring corresponding chemical index and monosaccharide composition.
Summary of the invention
In view of this, including the following steps the purpose of the present invention is to provide a kind of method for extraction and purification of anemaran：
1) pretreatment of rhizoma anemarrhenae obtains Common Anemarrhena Rhizome；
2) degreasing is carried out to Common Anemarrhena Rhizome using organic solvent；
3) it is extracted by water, alcohol precipitating is extracted and obtains thick anemaran；
4) thick anemaran purification：Low molecular weight impurities are gone in dialysis membrane dialysis, and DEAE- agarose Gel column adsorbing separation obtains
Anemaran desorbed solution is obtained, dialysis membrane, which is dialysed, is made anemaran purification component.
Preferably, in the method for extraction and purification of anemaran of the present invention, the Common Anemarrhena Rhizome is 40~60 mesh powder
End.
Preferably, in the method for extraction and purification of anemaran of the present invention, the organic solvent degreasing includes using
After alcohol degreasing, acetone degreasing is used；It is highly preferred that organic solvent degreasing of the present invention includes the following steps：
(1) alcohol degreasing：
Common Anemarrhena Rhizome is immersed in the ethyl alcohol that its weight is 10 times, mass concentration is 85%, is heated to reflux stirring at 70 DEG C
2h continues to stir 12h after cooling at room temperature, removes liquid；Secondary 10 times of its weight, the mass concentration of being added is 85% second
Alcohol stirs 5h at room temperature, removes liquid；
(2) acetone degreasing：
By the acetone soln that 5 times of weight is added in the rhizoma anemarrhenae powder after alcohol degreasing, mass concentration is 99%, after stirring 30 minutes
Liquid is removed, which is spontaneously dried at room temperature, obtains degreasing Common Anemarrhena Rhizome.
Preferably, in the method for extraction and purification of anemaran of the present invention, the step 3) passes through water extraction, alcohol precipitation
Extraction of forming sediment obtains thick anemaran and includes the following steps：
15-20 times of weight of water is added in the degreasing Common Anemarrhena Rhizome that step 2) is obtained, and stirring 2h is heated to reflux at 90 DEG C,
Centrifugation obtains extracting solution after cooling, and the extracting solution primary, merging obtains twice is extracted in residue continuation again in the same way, 50
The 1/3 of original volume is concentrated under the conditions of DEG C；Ethyl alcohol is added in the concentrate, concentration of alcohol is not less than 75%, adjusts temperature
To 4 DEG C, stand 12h, have polysaccharide floccule precipitation, filter out liquid, spontaneously dry at room temperature；Successively with the polysaccharide floccule
The dehydrated alcohol and 5 times of acetone that 5 times of weight wash respectively, and taking precipitate spontaneously dries, and thick anemaran is made.
Preferably, in the method for extraction and purification of anemaran of the present invention, the thick anemaran purification include with
Lower step：
A. the low molecular weight impurities of the thick anemaran are removed：
Thick anemaran made from step 3) is added to 10 times of weight of water, is dialysed and is separated with 3500Da dialysis membrane, is obtained
Molecular weight is greater than the dialyzate of 3500Da, and freeze-drying, which is made, removes impurity anemaran；
B. adsorbing separation：
The water that impurity anemaran addition weight is 0.04 times will be removed made from step a to dissolve, be heated to 60 DEG C of dissolutions, be used
The separation of DEAE- gelose gel column chromatography, obtains desorbed solution by elution；
C. purification obtains anemaran and refines component：
Step b resulting desorbed solution 3500Da dialysis membrane is dialysed, dialyzate of the molecular weight greater than 3500Da is taken to carry out
It is lyophilized to obtain the final product.
It is highly preferred that being washed in the step c using eluent in the method for extraction and purification of anemaran of the present invention
It takes off first to obtain just section desorbed solution using distillation water elution, then is eluted with NaCl and obtain secondary segment desorbed solution.
It is highly preferred that being washed in the step c using eluent in the method for extraction and purification of anemaran of the present invention
Take off for after first distilled water coutroi velocity 1.5mL/min being used to rinse 1h, collect desorbed solution 4h, obtain just section desorbed solution；0.5M is used again
NaCl with same flow velocity continue rinse 2h after, collect desorbed solution 3h；Improving parsing agent NaCl concentration is 1.0M, continues to collect parsing
Liquid 3h obtains secondary segment desorbed solution.
The present invention also provides the anemarans that the method for extraction and purification of above-mentioned anemaran obtains to refine component.
Compared with prior art, the present invention the present invention has the following advantages that：Present invention firstly provides anemaran purifications
The extracting method of component obtains anemaran by this method and refines component compared with conventional method obtains thick anemaran, tool
It is made of the chemical component and monosaccharide of different proportion, different molecular size ranges, and part purification component is shown to small
The higher activation capability of mouse macrophage RAW264.7, and it is stronger to the expression for promoting iNOS and relevant cell factor mRNA
Activation effect.
Detailed description of the invention
Fig. 1 be one embodiment of the present of invention in thick anemaran, refine component RI and UV map figure；
Fig. 2 be one embodiment of the present of invention in thick anemaran, purification component to macrophage proliferation influence diagram；
Fig. 3 be thick anemaran in one embodiment of the present of invention, purification component to RAW264.7 cell NO yield and
The influence of iNOS, mRNA expression of cytokines；
Fig. 4 is that the anemaran in one embodiment of the present of invention refines 2 activating macrophage of component generation cell factor
Situation schematic diagram.
Specific embodiment
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
The method for extraction and purification of 1 anemaran of embodiment
A, it pre-processes：After rhizoma anemarrhenae is removed impurity, 60 DEG C of drying are ground to 40 mesh powder；
B, degreasing：
(1) alcohol degreasing：
The Common Anemarrhena Rhizome of a step process is immersed in the ethyl alcohol that its weight is 10 times, mass concentration is 85%, is added at 70 DEG C
Hot return stirring 2h continues to stir 12h after cooling at room temperature, removes liquid；Secondary 10 times of its weight of addition, mass concentration are
85% ethyl alcohol stirs 5h at room temperature, removes liquid, the Common Anemarrhena Rhizome after obtaining alcohol degreasing；
(2) acetone degreasing：
By the acetone soln that 5 times of its weight is added in the Common Anemarrhena Rhizome after alcohol degreasing, mass concentration is 99%, 30 points are stirred
Liquid is removed after clock, which is spontaneously dried at room temperature, obtains degreasing Common Anemarrhena Rhizome；
C, Polyose extraction：
Degreasing Common Anemarrhena Rhizome made from b step is added to the water of 15-20 times of its weight, stirring is heated to reflux at 90 DEG C
2h is centrifuged after cooling and obtains extracting solution, and the extracting solution primary, merging obtains twice is extracted in residue continuation again in the same way,
The 1/3 of original volume is concentrated under the conditions of 50 DEG C to stop；Ethyl alcohol is added in the concentrate, until concentration of alcohol is not less than in its system
75%, adjustment temperature to 4 DEG C, stand 12h, have polysaccharide floccule precipitation, filter out liquid, spontaneously dry at room temperature；Successively use
5 times of dehydrated alcohol of polysaccharide floccule weight and 5 times of acetone wash respectively, and taking precipitate spontaneously dries, and it is more that thick rhizoma anemarrhenae is made
Sugar；
D, thick anemaran purification：
(1) low molecular weight impurities are gone：
Thick anemaran made from step c is added to the water of 10 times of its weight, is dialysed and is separated with 3500Da dialysis membrane, take it
Molecular weight is greater than the dialyzate of 3500Da, is lyophilized, and is made and removes impurity anemaran；
(2) adsorbing separation：
Will (1) step it is obtained except impurity anemaran be added the water dissolution that its weight is 0.04 times, be heated to 60 DEG C it is molten
Solution；With DEAE- gelose gel column chromatography (17-0709-01, GE Healthcare Bio-Science AB, Uppsala,
Sweden it) separates, after using distilled water coutroi velocity 1.5mL/min to rinse 1h first, collects desorbed solution 4h, be named as desorbed solution 1；
Again with 0.5M NaCl with same flow velocity continue rinse 2h after, collect desorbed solution 3h；Improving parsing agent NaCl concentration is 1.0M, after
It is continuous to collect desorbed solution 3h, it is named as desorbed solution 2；
(3) it refines：
The resulting desorbed solution 1 of (2) step and desorbed solution 2 are dialysed with 3500Da dialysis membrane respectively, its molecular weight is taken to be greater than
The dialyzate of 3500Da is lyophilized, and anemaran purification component 1 and purification component 2 is made.
(4) index determining：
It measures thick anemaran, purification component 1 and refines yield, chemical component and the monosaccharide composition of component 2 (referring to table
1), molecular weight (referring to Fig. 1 and table 2), measure bioactivity (referring to fig. 2), analyze purification component 2 on-link mode (OLM) (referring to
Table 3) and cytokine production (referring to Fig. 3).
The yield of the thick anemaran of table 1 and purification component, chemical component and monosaccharide composition
aYield=(thick anemaran weight/rhizoma anemarrhenae weight) × 100
bYield=(purification composition weight/injection splitter thick anemaran weight) × 100
n.d.cExpression is not detected
The thick anemaran extracted from rhizoma anemarrhenae is purified to obtain two components, is named as purification component 1, purification group
Divide 1, yield, chemical component and monosaccharide composition are shown in Table 1.Thick anemaran and the total sugar content of two components be 51.4%~
89.7%, protein content be 1.9%~14.5%, sulfate radical content be 3.4%~8.1%, glucuronic acid content be 1.0%~
30.2%.Monosaccharide composition is mainly mannose, the also glucose containing different content, arabinose, galactolipin, xylose and sandlwood
Sugar.The molecular characterization of anemaran is systematically analyzed using HPSEC-UV-MALLS-RI simultaneously, UV and RI chromatogram is shown in Fig. 1 (its
In, Figure 1A, B, C are respectively thick anemaran, purification component 1, UV the and RI chromatogram for refining component 2), molecular weight and revolution half
Diameter is shown in Table 2, it is known that thick anemaran and purification component 1 form (Peak I and Peak II), F by two peaks2It is made of a peak,
Molecular weight is 130.0 × 103~515.5 × 103U, the radius of gyration are 91.2~301.2nm.
Average molecular weight (the M of 2 anemaran of tablew), the radius of gyration (Rg)
The purification component effect test of 2 anemaran of embodiment
Analysis is handled as follows in thick anemaran that embodiment 1 obtains, purification component 1, purification component 2：
1) activating macrophage proliferative conditions measure
Cell Proliferation uses WST-1 (2- (4- iodobenzene) -3- (4- nitrobenzene) -5- (2,4- disulfobenzene) -2H- tetrazole
Sodium salt) cell Proliferation and citotoxicity detection kit (EZ-cytox, Daeillab service CO., LTD, Korea), tool
Body method is as follows：100 μ L RAW264.7 cells (1 × 106/mL) (ATCC) are added in 96 microwell plates, train in 37 DEG C of 5%CO2
Preculture in case (Excella ECO-170, New Brunswick Scientific, Scotland) is supported for 24 hours, to discard supernatant
The sample solution of 200 μ L various concentrations is added in cell for liquid, and culture solution is as blank group.Culture discards supernatant liquid afterwards for 24 hours, carefully
110 μ L volume fraction 10%WST-1 solution are added in born of the same parents, continuation cultivates 1h in the incubator, uses microplate reader under 450nm wavelength
Light absorption value is measured under (EL-800, BioTek instruments, Winooski, VT, USA), cell proliferation rate calculation formula is such as
Under：
As shown in Figure 4, it is known that the thick anemaran and refine component 1, purification component 2 in macrophage that embodiment 1 obtains
Proliferation rate influence above 100%, illustrate there is no toxicity to macrophage, and cell Proliferation can be promoted.
2) polysaccharide activating macrophage generates determination of nitric oxide and RT-PCR detects mRNA expression of cytokines
A. polysaccharide activating macrophage generates determination of nitric oxide
The NO yield of sample is measured using macrophage supernatant, and specific method is that 100 μ L RAW264.7 are thin
Born of the same parents (1 × 106A/mL) it is added in 96 microwell plates, contain 5%CO at 37 DEG C2Incubator in preculture for 24 hours, pour out supernatant,
The test sample solution (2,5 and 10 μ g/mL) of 200 μ L various concentrations is added, culture solution is made as negative control, 1 μ g/mL LPS
For positive control.Culture draws 100 μ L supernatants afterwards for 24 hours and 100 μ L Griess reagents is added, after being protected from light 10min at room temperature
Absorbance is measured with microplate reader under 540nm wavelength, NO yield is calculated using sodium nitrite as standard curve.
B.RT-PCR detects mRNA expression of cytokines
1mL RAW264.7 cell (1 × 106A/mL) it is added in 24 orifice plates, containing 37 DEG C in 5%CO2 incubator
Preculture for 24 hours, discards supernatant liquid, and sample solution (2,5 and of 1mL LPS (2 μ g/mL) and various concentration are separately added into cell
10 μ g/mL) culture 18h.Cracking is carried out to cell with Trizol (Invitrogen, Carlsbad, CA, USA) later to extract always
RNA, using micro-spectrophotometer (MS-1000, Tech&innovation, China) measure RNA concentration, adjust concentration after with
RNA synthesizes cDNA through reverse transcription for template, then upstream and downstream primer is added by template of cDNA.The product of amplification shines after electrophoresis
Phase, obtained band are analyzed using Quantity One 1D Analysis Software, and same β-actin is carried out later
Comparison obtains the relative concentration of mRNA expression of cytokines.
As a result as follows：
Analyze anemaran activation RAW264.7 cell simultaneously and generate NO ability, as a result see Fig. 3 A, thick anemaran and
Two purification components can stimulate RAW264.7 cell to generate certain density NO, and in the concentration range of 2,5,10 μ g/mL
Concentration dependent is inside shown, purification component 1, purification component 2 are then shown to the highest activation capability of RAW264.7 cell.Slightly
Anemaran and two purification components can promote the expression (Fig. 3 B and 3C) of iNOS and relevant cell factor mRNA, with other groups
Split-phase ratio, purification component 1, purification 2 processing group of component show strongest activation effect (referring to fig. 4, wherein A be each processing
To the TNF-α of RAW264.7 cell, COX-2, the electrophoretogram of IL-1 cytokine-expressing；B is that each processing is thin to RAW264.7
The TNF-α mRNA of born of the same parents expresses relative concentration；C is that each processing expresses relative concentration to the COX-2mRNA of RAW264.7 cell；D
Relative concentration is expressed for IL-6mRNA of each processing to RAW264.7 cell).
From the foregoing, it will be observed that purification component 2 shows strongest bioactivity, so analyzing its on-link mode (OLM), as a result
It is shown in Table 3.Know that its main chain is made of 1 → 4 link mannose residue, simultaneously containing a small amount of other residues.
3 anemaran of table refines 2 methylation analysis results of component
| Appearance number | Glucosides key type | Peak area (%) |
| 1 | Ara f-(1→ | 12.6 |
| 2 | Xyl f-(1→ | 2.7 |
| 3 | →4)-Ara-(1→ | 5.6 |
| 4 | Glu-(1→ | 1.6 |
| 5 | Gal/-(1→ | 3.8 |
| 6 | →2)-Gal/-(1→ | 2.3 |
| 7 | →4)-Glu-(1→ | 0.4 |
| 8 | →4)-Man/(1→ | 51.2 |
| 9 | →3)Gal-(1→ | 4.6 |
| 10 | →3)-Gal/-(1→ | 2.8 |
| 11 | →6)-Gal/-(1→ | 1.5 |
| 12 | →3,4)-Gal/-(1→ | 0.7 |
| 13 | →2,6)-Gal/-(1→ | 0.4 |
| 14 | →3,6)-Glu-(1→ | 6.4 |
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of method for extraction and purification of anemaran, includes the following steps：
1) pretreatment of rhizoma anemarrhenae obtains Common Anemarrhena Rhizome；
2) degreasing is carried out to Common Anemarrhena Rhizome using organic solvent；
3) it is extracted by water, alcohol precipitating is extracted and obtains thick anemaran；
4) thick anemaran purification：Low molecular weight impurities are gone in dialysis membrane dialysis, and DEAE- agarose Gel column adsorbing separation is known
Female polysaccharide desorbed solution, dialysis membrane, which is dialysed, is made anemaran purification component.
2. the method for extraction and purification of anemaran according to claim 1, which is characterized in that the Common Anemarrhena Rhizome be 40~
60 mesh powder.
3. the method for extraction and purification of anemaran according to claim 1, which is characterized in that the organic solvent degreasing packet
It includes using after alcohol degreasing, uses acetone degreasing.
4. the method for extraction and purification of anemaran according to claim 3, which is characterized in that the organic solvent degreasing packet
It includes：
(1) alcohol degreasing：
Common Anemarrhena Rhizome is immersed in the ethyl alcohol that its weight is 10 times, mass concentration is 85%, stirring 2h, warp are heated to reflux at 70 DEG C
Continue to stir 12h at room temperature after cooling, removes liquid；Secondary 10 times of its weight, the mass concentration of being added is 85% ethyl alcohol, room temperature
Lower stirring 5h removes liquid；
(2) acetone degreasing：
By the acetone soln that 5 times of weight is added in the rhizoma anemarrhenae powder after alcohol degreasing, mass concentration is 99%, stirring removes after 30 minutes
Liquid spontaneously dries the powder at room temperature, obtains degreasing Common Anemarrhena Rhizome.
5. the method for extraction and purification of anemaran according to claim 1, which is characterized in that the step 3) is mentioned by water
It takes, alcohol precipitating is extracted the thick anemaran of acquisition and included the following steps：
15-20 times of weight of water is added in the degreasing Common Anemarrhena Rhizome that step 2) is obtained, and stirring 2h is heated to reflux at 90 DEG C, cooling
Centrifugation obtains extracting solution afterwards, and the extracting solution primary, merging obtains twice is extracted in residue continuation again in the same way, in 50 DEG C of items
The 1/3 of original volume is concentrated under part；Ethyl alcohol is added in the concentrate, concentration of alcohol is not less than 75%, adjustment temperature to 4
DEG C, stand 12h, have polysaccharide floccule precipitation, filter out liquid, spontaneously dry at room temperature；Successively with the polysaccharide floccule weight
5 times of dehydrated alcohol of amount and 5 times of 99% acetone of mass concentration wash respectively, and taking precipitate spontaneously dries, and it is more that thick rhizoma anemarrhenae is made
Sugar.
6. the method for extraction and purification of anemaran according to claim 1, which is characterized in that the thick anemaran purification
Include the following steps：
A. the low molecular weight impurities of the thick anemaran are removed：
Thick anemaran made from step 3) is added to 10 times of weight of water, is dialysed and is separated with 3500Da dialysis membrane, obtains molecule
Amount is greater than the dialyzate of 3500Da, and freeze-drying, which is made, removes impurity anemaran；
B. adsorbing separation：
The water that impurity anemaran addition weight is 0.04 times will be removed made from step a to dissolve, be heated to 60 DEG C of dissolutions, be used
The separation of DEAE- gelose gel column chromatography, obtains desorbed solution by elution；
C. purification obtains anemaran and refines component：
Step b resulting desorbed solution 3500Da dialysis membrane is dialysed, dialyzate of the molecular weight greater than 3500Da is taken to be lyophilized
To obtain the final product.
7. the method for extraction and purification of anemaran according to claim 6, which is characterized in that using washing in the step c
De- liquid elution elutes acquisition secondary segment desorbed solution first to use distillation water elution to obtain just section desorbed solution, then with NaCl.
8. the method for extraction and purification of anemaran according to claim 6, which is characterized in that using washing in the step c
De- liquid elution is after first distilled water coutroi velocity 1.5mL/min being used to rinse 1h, collects desorbed solution 4h, obtains first section desorbed solution；Again
With 0.5M NaCl with same flow velocity continue rinse 2h after, collect desorbed solution 3h；Improving parsing agent NaCl concentration is 1.0M, is continued
Desorbed solution 3h is collected, secondary segment desorbed solution is obtained.
9. the anemaran purification that the method for extraction and purification of anemaran described in any one obtains according to claim 1~8
Component.
Priority Applications (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110051683A (en) * | 2019-05-06 | 2019-07-26 | 广州中医药大学(广州中医药研究院) | Application of the anemaran in the drug of preparation treatment scheroma |
| CN110540603A (en) * | 2019-08-30 | 2019-12-06 | 南开大学 | Rhizoma anemarrhenae polysaccharide, and preparation method, identification method and application thereof |
| CN112876577A (en) * | 2021-03-18 | 2021-06-01 | 华南理工大学 | Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof |
| CN115141288A (en) * | 2022-07-27 | 2022-10-04 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
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| CN102166294A (en) * | 2011-04-08 | 2011-08-31 | 匡海学 | Common anemarrhena polysaccharide extractive and preparation method and medicinal purposes thereof |
| CN105061630A (en) * | 2015-09-18 | 2015-11-18 | 黑龙江八一农垦大学 | Method for extracting and purifying gentiana polysaccharide |
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| CN102166294A (en) * | 2011-04-08 | 2011-08-31 | 匡海学 | Common anemarrhena polysaccharide extractive and preparation method and medicinal purposes thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110051683A (en) * | 2019-05-06 | 2019-07-26 | 广州中医药大学(广州中医药研究院) | Application of the anemaran in the drug of preparation treatment scheroma |
| CN110051683B (en) * | 2019-05-06 | 2021-07-27 | 广州中医药大学(广州中医药研究院) | Application of rhizoma anemarrhenae polysaccharide in preparing medicine for treating xerophthalmia |
| CN110540603A (en) * | 2019-08-30 | 2019-12-06 | 南开大学 | Rhizoma anemarrhenae polysaccharide, and preparation method, identification method and application thereof |
| CN110540603B (en) * | 2019-08-30 | 2021-12-24 | 南开大学 | Rhizoma anemarrhenae polysaccharide, and preparation method, identification method and application thereof |
| CN112876577A (en) * | 2021-03-18 | 2021-06-01 | 华南理工大学 | Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof |
| CN112876577B (en) * | 2021-03-18 | 2022-02-22 | 华南理工大学 | Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof |
| CN115141288A (en) * | 2022-07-27 | 2022-10-04 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
| CN115141288B (en) * | 2022-07-27 | 2023-09-08 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
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