CN104177510A - Alpha-1,4-glucan and its preparation method and use - Google Patents
Alpha-1,4-glucan and its preparation method and use Download PDFInfo
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- CN104177510A CN104177510A CN201310190825.XA CN201310190825A CN104177510A CN 104177510 A CN104177510 A CN 104177510A CN 201310190825 A CN201310190825 A CN 201310190825A CN 104177510 A CN104177510 A CN 104177510A
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- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
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Abstract
The invention relates to a polysaccharide extracted from honeysuckle flower and its preparation method and use in preparation of drugs for treating Alzheimer's disease. Concretely, the invention relates to alpha-1,4-glucan extracted from honeysuckle flower. The preparation method comprises the following steps of extracting crude polysaccharide from honeysuckle flower and carrying out alcohol precipitation, protein removal and multiple types of column chromatography purification processes to obtain the alpha-1,4-glucan. An in-vitro experiment proves that the alpha-1,4-glucan can substantially inhibit aggregation of a key Alzheimer's disease-causing factor A beta 42 and can inhibit A beta 42 aggregation-induced nerve cell toxicity and thus the alpha-1,4-glucan can be used as a latent polysaccharide drug for treating Alzheimer's disease.
Description
Technical field
The present invention relates to Chinese herbal medicine extracting polysaccharide, more particularly, relate to a kind of from Japanese Honeysuckle (Flos Lonicerae), extract α-Isosorbide-5-Nitrae-dextran of obtaining, its preparation method with and treat the application in the medicine of alzheimer's disease in preparation.
Background technology
Along with the deep development of carbohydrate chemistry and glycobiology, vegetable polysaccharides is as the important biologically active substance of a class, be proved the multiple biological activity such as there is immunomodulatory, antitumor, antiviral, anti-oxidant and anti-infective by large quantity research, and little to the toxic side effect of body.Therefore, having bioactive polysaccharide comes into one's own day by day.
At present, become gradually the advanced subject of domestic and international the world of medicine taking carbohydrate as basic drug research and development.And the polysaccharide material of glucide, especially resources of Chinese medicinal herb is extensive based on biological function, the many advantages such as toxic side effect is little, aboundresources, are also subject to various countries research staff's very big attention, have potential application prospect widely.
Japanese Honeysuckle, as a kind of important traditional Chinese medicine, is the dry flower of climbing plant honeysuckle, records according to Shennong's Herbal, it is cold in nature, taste is sweet, has effect clearing heat and detoxicating, wind-dispelling heat-dissipating, is mainly used in clinically treating the swollen furuncle poison of common cold due to wind-heat, carbuncle, toxic-heat and blood stasis, anal fistula and has blood in stool etc.Japanese Honeysuckle is rich in various active composition and composition complexity, pharmacological research show these compounds have broad-spectrum antimicrobial, antiviral, antitumor, strengthen the multiple biological activitys such as immunity and antipyretic and anti-inflammatory, hepatic cholagogic, antiulcer agent.
Polysaccharide material, as the important component part of Japanese Honeysuckle drug effect, is studied less at present.Existing several researchs for Flos Lonicerae polysaccharide only limit to the extraction to its Crude polysaccharides
[1-3]and the preliminary screening of antibacterial antioxidation biology activity etc.
[4-6].The effect of Flos Lonicerae polysaccharide in treatment nervous system disorders, there is no report.Alzheimer's disease (Alzheimer ' s disease, AD) is also called presenile dementia, is a kind of nerve degenerative diseases of chronic progressive external, and main manifestations is that gradual memory capability declines, cognition dysfunction and lose the independent self-care ability of life.Along with the continuous aggravation of aging population, the sickness rate of AD also raises year by year, has become one of health problem of most important public attention
[7].
The unconventionality expression of amyloid beta (β-amyloid protein, A β) in brain and deposition are to think at present to cause the core link of AD
[8].In AD patient, due to factors such as AD associated gene mutation, concentration of metal ions increase and pH changes, cause microenvironment in brain to change, thereby the conformation of initiation amyloid change, change into and be easy to be gathered into fibrous beta sheet by alpha-helix, thereby can disturb Ca
2+stable state, initiated oxidation stress, mitochondria dysfunction, inflammatory reaction, induction Tau albumen Hyperphosphorylationof and cause the cascade neurotoxicities such as neuron loss, and finally cause dementia
[9].Therefore,, based on the neurotoxicity of A β, taking A β as action target spot, finding the medicine that reduces A β formation, inhibition A beta peptide aggregation and accelerate A β degraded is the study hotspot for the treatment of at present AD medicine
[10].
We test discovery, and the polysaccharide in Flos lonicerae source can significantly suppress A β in vitro
42assemble, and suppress A β in people's neuroma parent cell level
42the cytotoxicity of aggregation inducing, has the effect of potential treatment alzheimer's disease.
[1] Zhang Yu, horsepower, Chen Wen. alcohol bleed formulation is extracted Flos Lonicerae polysaccharide [J]. medical Leader, 2006,25 (11): 1118-1120.
[2] Deng Qinghua. with orthogonal experiment optimizing metal honeysuckle flower polysaccharide extracting process [D]. Changchun: Northeast Normal University, 2008.
[3] Zhao Peng, Li Wenhong, Zhu is sea suddenly, etc. response surface method is optimized Flos Lonicerae polysaccharide ultrasonic extraction process research [J]. Food science, 2009,30 (20): 151-154
[4] Li Erchun. the separation and purification of Flos Lonicerae polysaccharide and bioactivity research [D]. Xi'an: Shaanxi Normal University, 2009.
[5] Lin Xiongping, Chen Xiaoqing, Su Yucai, etc. Japanese Honeysuckle and Leaf of Chinese Holly polyoses extract anti-microbial activity research [J]. subtropical plant science, 2008,37 (1): 51-53.
[6] Yin Hongmei, Lv Xinyong, Xiao Wei. the optimum preparation condition of Flos Lonicerae polysaccharide and immunocompetence research [J]. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2010,35 (4): 453-455.
[7]Goedert?M,Spillantinim?G.A?century?of?Alzheimers'disease[J].Science,2006,314(5800):777-781
[8]Hardy?J,Selkoe?D.J.The?amyloid?hypothesis?of?Alzheimer’s?disease:progress?and?problems?on?the?road?to?therapeutics[J].Science,2002(297):353-356.
[9]Cappai?R.Barnham?K.J."Delineating?the?Mechanism?of?Alzheimer’s?Disease?Aβ?Peptide?Neurotoxicity."Neurochemical?Research[J].2007,33(3):526-532.
[10]Francesca?M,Alina?S,Bengt?W,Patrizia?M,Miia?K.Alzheimer’s?disease:clinical?trials?and?drug?development[J].Lancet?Neurol,2010(9):702-716.
Summary of the invention
The present invention utilizes a kind of simple and effective polysaccharide extracting process and method, taking Japanese Honeysuckle as raw material has obtained a kind of α-Isosorbide-5-Nitrae-dextran, pharmacological evaluation shows, described α-Isosorbide-5-Nitrae-dextran, under the concentration of 100 μ g/ml, can suppress A β in vitro completely
42gathering, and on cell levels, suppress A β
42assemble the cytotoxicity causing, therefore, described α-Isosorbide-5-Nitrae-dextran is expected to exploitation becomes a kind of carbohydrate medicine for the treatment of alzheimer's disease.
One object of the present invention is to provide a kind of α-Isosorbide-5-Nitrae-dextran, and its structural formula is as follows:
Wherein, x and y are integer and x+y=14;
N is positive integer;
Weight average molecular weight range is: about 10-100kDa.
The weight-average molecular weight of described α-Isosorbide-5-Nitrae-dextran is preferably about 15-80kDa, more preferably 20-50kDa.
Another object of the present invention is to provide a kind of method of preparing described α-Isosorbide-5-Nitrae-dextran taking Japanese Honeysuckle as raw material.
Said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through alcohol degreasing, water extraction, filtration, and gained filtrate is concentrated, then through 15% trichoroacetic acid(TCA) deproteinated, and neutralization, dialysis, concentrated, alcohol precipitation, centrifugal, vacuum-drying, obtain water extraction Japanese Honeysuckle Crude polysaccharides;
B. polysaccharide purification: described water extraction Japanese Honeysuckle Crude polysaccharides is first carried out to preliminary classification with DEAE Mierocrystalline cellulose anion column, and water elution obtains neutral polysaccharide component, and then uses gel chromatography column purification, obtains α-Isosorbide-5-Nitrae-dextran.
Preferably, said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through 75%-95% alcohol degreasing, dry, adds deionized water, under heating condition, extract, filter, residue is used deionized water extraction again, so repeatedly extract 2-6 time, filtrate merges, and heating is concentrated, the trichoroacetic acid(TCA) that concentrated solution is 15% through final concentration is deproteinated at 4 DEG C, centrifugal, and supernatant liquor is through neutralization, dialysis, reconcentration, adds 3 times to the 75%-95% of concentrated solution volume ethanol, centrifugal must precipitation, precipitation obtains water extraction Japanese Honeysuckle Crude polysaccharides through vacuum-drying;
B. polysaccharide purification: get described Japanese Honeysuckle Crude polysaccharides, water dissolution, centrifugal, supernatant liquor separates by DEAE Mierocrystalline cellulose anion column, with distilled water wash-out, sulfuric acid-phynol detects, collect merging elutriant, concentrated frozen is dried to obtain water elution component, and then adopts G150 gel chromatographic columns to separate, purifying obtains α-Isosorbide-5-Nitrae-dextran.
More preferably, said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through 95% alcohol degreasing 7-10 days, natural drying at room temperature, and dried Japanese Honeysuckle adds the deionized water of 20 times of weight, at 100 DEG C, extract 2-6 time, each 5-7h, filtrate merges, heating is concentrated, and the trichoroacetic acid(TCA) that concentrated solution is 15% through final concentration is deproteinated at 0-4 DEG C, centrifugal, supernatant liquor is through neutralization, dialysis, reconcentration, adds 3 times to 95% ethanol of concentrated solution volume, centrifugal must precipitation, precipitation obtains water extraction Japanese Honeysuckle Crude polysaccharides through vacuum-drying;
B. polysaccharide purification: get described Japanese Honeysuckle Crude polysaccharides, add in the water of 10 times of weight and dissolve, centrifugal, supernatant liquor separates by DEAE Mierocrystalline cellulose anion column, with distilled water wash-out, sulfuric acid-phynol detects, and collects merging elutriant, and concentrated frozen is dried to obtain water elution component, this water elution component is dissolved in to 0-0.2mol/L NaCl, after centrifugal, separate by G150 gel chromatographic columns, purifying obtains α-Isosorbide-5-Nitrae-dextran.
Polysaccharide structures qualification:
Measure through High Performance Gel Permeation Chromatography (HPGPC), be about 10-100kDa according to the weight average molecular weight range of α-Isosorbide-5-Nitrae-dextran of the present invention.Carried out sugared compositional analysis, sent into gas chromatograph analysis by polysaccharide complete hydrolysis, reduction, acetylize, extraction, after concentrated.Sugar compositional analysis result shows, only contains glucose unit according to α-Isosorbide-5-Nitrae-dextran of the present invention.Then react to polysaccharide exhaustive methylation with methyl iodide, then acid hydrolysis completely, reduction, acetylize, extraction, concentrated rear use gas chromatograph and spectrometer analysis.In conjunction with infrared and nuclear magnetic resonance spectroscopy (referring to Fig. 2 and 3) and the result that methylates, determine according to α-1 of the present invention, 4-dextran is with α-1,4 glucose that connect are main chain, and in C6 position with a small amount of α-1, the 4 dextran branched structures that connect, average every 16 glucosyl residues contain 1 side chain.
Another object of the present invention is to provide α-Isosorbide-5-Nitrae-dextran according to the present invention in the purposes of preparing in the medicine for the treatment of the nervous system injury of being induced by amyloid-beta.Preferably, the invention provides the purposes of α-Isosorbide-5-Nitrae-dextran according to the present invention in the medicine of preparation treatment alzheimer's disease.
The present invention is further elaborated by the following drawings and embodiment, but does not limit content of the present invention.
Brief description of the drawings:
Fig. 1 is the Purity figure of α-Isosorbide-5-Nitrae-dextran LJW0F2 of embodiment 1;
Fig. 2 is the infrared spectrogram (infrared spectrogram, IR) of α-Isosorbide-5-Nitrae-dextran LJW0F2 of embodiment 1;
Fig. 3 is α-Isosorbide-5-Nitrae-dextran LJW0F2 of embodiment 1
13c NMR spectrogram;
Fig. 4 is that α-Isosorbide-5-Nitrae-dextran LJW0F2 of embodiment 1 suppresses A β
42aggregation activity measurement result; (Fig. 4 A is thioflavin fluoroscopic examination result; Fig. 4 B is A β
42hatch separately atomic force microscope detected result after 7 days; And Fig. 4 C is LJW0F2 and A β
42hatch altogether atomic force microscope detected result after 7 days)
Fig. 5 is that α-Isosorbide-5-Nitrae-dextran LJW0F2 of embodiment 1 suppresses A β
42the neuronal cell toxicity result of aggregation inducing.
Embodiment
Embodiment 1: the preparation of α-Isosorbide-5-Nitrae-dextran LJW0F2
A. polysaccharide extracts:
Dry Japanese Honeysuckle (place of production is Pingyi county, supports and hall medicine company chain operation company limited purchased from Shanghai), the alcohol degreasing with 95% one week, then natural drying at room temperature.Dried Japanese Honeysuckle 1000g extracts 5 times with boiling water 20L, each 6h.Sulfuric acid-phynol detects extremely without significant reaction, filter, each extracting solution is merged to post-heating and be concentrated into 3L, after cooling, adding about 500g trichoroacetic acid(TCA) to make its ultimate density is 15%, deproteinated at 4 DEG C, by centrifugal, it is 7.0 that supernatant liquor is neutralized to pH value through 1mol/L NaOH, then flow water dialysis 72h, in dialysis tubing, liquid is concentrated into 2L volume, under agitation add 95% long-pending ethanol of triploid, hold over night, supernatant liquor inclines, centrifugation, the absolute ethanol washing of 2 times of volumes for gained precipitation, centrifugation, precipitation is put vacuum-drying at 40 DEG C, obtain water extraction Japanese Honeysuckle Crude polysaccharides LJW75g.
B. polysaccharide purification:
Get the Japanese Honeysuckle Crude polysaccharides LJW10g of above-mentioned preparation, 100mL water dissolution, the centrifugal insolubles of removing, supernatant liquor passes through Cl
-type DEAE-cellulose column (GE Lifescience company) carries out initial gross separation.With distilled water wash-out, sulfuric acid-phynol method is drawn elution curve, collects merging elutriant according to elution curve.Elutriant obtains polysaccharide LJW02.4g after concentrated and lyophilize.Get LJW0200mg and add 2mL0.2M NaCl dissolving centrifugal, supernatant liquor carries out purifying by G150 gel chromatographic columns (GE Lifescience company), flow velocity is 0.3mL/min, sulfuric acid-phynol method is drawn elution curve, collect respectively merging elutriant according to elution curve, purifying obtains polysaccharide fraction LJW0F2100mg so repeatedly.
EXPERIMENTAL EXAMPLE 1: polysaccharide structures is resolved:
Show that through efficient gel chromatography (HPGPC) analysis the relative weight-average molecular weight of LJW0F2 is about 37.1kDa, its purity testing figure is shown in Fig. 1.Sugar compositional analysis shows that LJW0F2 is a dextran.Infared spectrum shows, 3403cm
-1for O-H stretching vibration absorption peak, 2927cm
-1for C-H stretching vibration absorption peak, 1000-1400cm
-1neighbouring is C-O and sugared ring vibration signal, 1720cm
-1near there is no absorption peak, show that this polysaccharide does not contain uronic acid (Fig. 2).
13in C NMR spectrum, being positioned at the carbon signal of δ 100.98, is the C-1 signal of alpha-glucan.Other carbon signals are followed successively by C-2 (δ 72.97), C-3 (δ 74.60), and C-4 (δ 78.08), C-5 (δ 72.61) and C6 (δ 61.70) are (Fig. 3).
In polysaccharide LJW0F2, the kind of saccharide residue mode of connection and ratio can be used methylation analysis.Result shows, the glucosyl residue of LJW0F2 has three kinds of mode of connection, be respectively Isosorbide-5-Nitrae-, Isosorbide-5-Nitrae, 6-and end connect glucosyl group, its ratio is 14:1:1.Can find from aforementioned proportion, the backbone structure of LJW0F2 should be the glucan structure of Isosorbide-5-Nitrae-connection, and has the dextran branched structure of Isosorbide-5-Nitrae-connection at the part C6 of main chain bit strip.
Above result shows that the structure of LJW0F2 is:
Wherein, x and y are integer and x+y=14;
EXPERIMENTAL EXAMPLE 2
Suppress A β
42aggregation Test
1) thioflavin fluorescent mark experiment
By A β
42powder (Rpeptide company) is dissolved in (concentration is 2mM) in the anhydrous DMSO of 110 μ L, is made into mother liquor.Get 1 this solution of μ L and be dissolved in 19 μ L phosphoric acid buffer (50mM phosphoric acid salt, pH7.5,100mM NaCl, 0.02%NaN
3) in, or 1 this solution of μ L add the LJW0F2 polysaccharide soln (0.5mg/mL, 1.0mg/mL, 2.0mg/mL) of 10 μ L different concns to add again 9 μ L PBS damping fluids, hatch altogether 30min for 37 DEG C, add thioflavin solution (6.25 μ M thioflavins are dissolved in 50mM glycine-NaOH, pH8.5) 80 μ L, hatch altogether for 37 DEG C, detect (Novostar every 2h by microplate reader, BMG labtech company), detection wavelength is Ex=450/10nm, Em=483/10nm.
From Fig. 4 A, α-Isosorbide-5-Nitrae-dextran LJW0F2, under the final concentration of 100 μ g/mL, can suppress A β completely
42gathering.
2) atomic force microscope test experience
For further verifying that polysaccharide LJW0F2 suppresses A β
42the result of assembling, adopts atomic force microscope observation polysaccharide LJW0F2 to A β
42the impact of accumulation shape.The A β that is 2mM by 1 μ L concentration
42, 10 μ L concentration are 1.0mg/mL LJW0F2 polysaccharide soln is dissolved in 89 μ L distilled waters, hatch altogether 7 days for 37 DEG C.The A β that is 2mM by 1 μ L concentration
42be dissolved in 99 μ L Millipore water, or the LJW0F2 polysaccharide soln that is 1.0mg/mL by 10 μ L concentration is dissolved in 90 μ L Millipore water, hatches 7 days for 37 DEG C, in contrast.Get 5 μ L sample solution is dropped on clean sheet mica and carefully dried up, under atomic force microscope, under the pattern of rapping of (Nanoscope III a, Veeco Instrucments company), test.
Be can be observed A β by Fig. 4 B
42hatching separately after 7 days, occurring a large amount of oligomer, and hatch altogether (Fig. 4 C) after 7 days with polysaccharide LJW0F2, do not finding significantly to assemble.This result and thioflavin fluoroscopic examination result match, and prove that polysaccharide has the A of inhibition β
42the activity of assembling.
EXPERIMENTAL EXAMPLE 3:
α-Isosorbide-5-Nitrae-dextran LJW0F2 suppresses A β
42assemble the neuronal cell toxicity test causing
1) cell cultures
Human neuroblastoma cell SH-SY5Y(is purchased from Chinese Academy of Sciences's cell bank) with the MEM of volume ratio 1:1 and Ham ' s F12 substratum (containing 10% foetal calf serum) in 5%CO
237 DEG C of cultivations of incubator, change liquid for 2-3 days 1 time.After cell attachment covers with, with 0.25% trypsin Invitrogen company) digestion after, be inoculated into 96 porocyte culture plates with 35000 cells/well, every pore volume 100 μ L.Cultivate 16h, make cell attachment for 37 DEG C.
α-Isosorbide-5-Nitrae-dextran LJW0F2 solution (0,1000,100,10 μ g/mL) of different concns is with A β
42(200 μ M) or isopyknic DMSO, 37 DEG C of water-baths were hatched after 4 days, sucked overnight incubation cell conditioned medium, added the above-mentioned solution of hatching 4 days with substratum dilution, and every hole 100 μ L, make A β
42final concentration is 2 μ M, and α-Isosorbide-5-Nitrae-dextran LJW0F2 final concentration is (0,10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL).37 DEG C are continued to cultivate 48h, and CCK-8 measures cell survival rate.
2) cell counting test kit (Cell Counting Kit-8, CCK-8) is measured
Every hole adds CCK-8 solution 10 μ L.Continue to hatch 4h, select 450nm wavelength, on enzyme linked immunological monitor, (Novostar, BMG labtech company) measures each hole absorbance value, records result, calculates cell survival rate.
As shown in Figure 5,1) A β
42hatch separately 4 days, add after SH-SY5Y cell, this cell survival rate compared with normal group 10% left and right that declines, illustrates A β
42oligomer has produced cytotoxicity to SH-SY5Y cell.2) as A β
42hatch altogether 4 days with different concns α-Isosorbide-5-Nitrae-dextran LJW0F2, add after SH-SY5Y cell, the survival rate of this cell returns to normal level, illustrates that α-Isosorbide-5-Nitrae-dextran LJW0F2 can suppress A β
42the neurotoxicity of aggregation inducing.And α-Isosorbide-5-Nitrae-dextran LJW0F2 adds separately SH-SY5Y cell, surviving of this cell had no significant effect.These results show, α-Isosorbide-5-Nitrae-dextran LJW0F2 can be by suppressing A β
42oligomerization so that protect its damage to neuronal cell SH-SY5Y.
Claims (7)
1. α-Isosorbide-5-Nitrae-dextran, its structural formula is as follows:
Wherein, x and y are integer and x+y=14;
N is positive integer;
The weight average molecular weight range of described α-Isosorbide-5-Nitrae-dextran is: 10-100kDa.
2. α-Isosorbide-5-Nitrae-dextran according to claim 1, its weight average molecular weight range is 15 to 80kDa.
3. a method of preparing α-Isosorbide-5-Nitrae-dextran according to claim 1 taking Japanese Honeysuckle as raw material, said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through alcohol degreasing, water extraction, filtration, and gained filtrate is concentrated, then through 15% trichoroacetic acid(TCA) deproteinated, and neutralization, dialysis, concentrated, alcohol precipitation, centrifugal, vacuum-drying, obtain water extraction Japanese Honeysuckle Crude polysaccharides;
B. polysaccharide purification: described water extraction Japanese Honeysuckle Crude polysaccharides is first carried out to preliminary classification with DEAE Mierocrystalline cellulose anion column, and water elution obtains neutral polysaccharide component, and then uses gel chromatography column purification, obtains α-Isosorbide-5-Nitrae-dextran.
4. method according to claim 3, said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through 75%-95% alcohol degreasing, dry, adds deionized water, under heating condition, extract, filter, residue is used deionized water extraction again, so repeatedly extract 2-6 time, filtrate merges, and heating is concentrated, the trichoroacetic acid(TCA) that concentrated solution is 15% through final concentration is deproteinated at 4 DEG C, centrifugal, and supernatant liquor is through neutralization, dialysis, reconcentration, adds 3 times to the 75%-95% of concentrated solution volume ethanol, centrifugal must precipitation, precipitation obtains water extraction Japanese Honeysuckle Crude polysaccharides through vacuum-drying;
B. polysaccharide purification: extracting honeysuckle Crude polysaccharides, water dissolution, centrifugal, supernatant liquor separates by DEAE Mierocrystalline cellulose anion column, with distilled water wash-out, sulfuric acid-phynol detects, collect merging elutriant, concentrated frozen is dried to obtain water elution component, and then adopts G150 gel chromatographic columns to separate, purifying obtains α-Isosorbide-5-Nitrae-dextran.
5. method according to claim 4, said method comprising the steps of:
A. polysaccharide extracts: dry Japanese Honeysuckle is through 95% alcohol degreasing 7-10 days, natural drying at room temperature, and dried Japanese Honeysuckle adds the deionized water of 20 times of weight, at 100 DEG C, extract 2-6 time, each 5-7h, filtrate merges, heating is concentrated, and the trichoroacetic acid(TCA) that concentrated solution is 15% through final concentration is deproteinated at 0-4 DEG C, centrifugal, supernatant liquor is through neutralization, dialysis, reconcentration, adds 3 times to 95% ethanol of concentrated solution volume, centrifugal must precipitation, precipitation obtains water extraction Japanese Honeysuckle Crude polysaccharides through vacuum-drying;
B. polysaccharide purification: extracting honeysuckle Crude polysaccharides, add in the water of 10 times of weight and dissolve, centrifugal, supernatant liquor separates by DEAE Mierocrystalline cellulose anion column, with distilled water wash-out, sulfuric acid-phynol detects, and collects merging elutriant, and concentrated frozen is dried to obtain water elution component, water elution component is dissolved in to 0-0.2mol/LNaCl solution, after centrifugal, separate by G150 gel chromatographic columns, purifying obtains α-Isosorbide-5-Nitrae-dextran.
6. the purposes in the medicine of the nervous system injury that α-Isosorbide-5-Nitrae-dextran according to claim 1 is induced by amyloid-beta in preparation treatment.
7. purposes according to claim 6, wherein, described nervous system injury of being induced by amyloid-beta is alzheimer's disease.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310190825.XA CN104177510B (en) | 2013-05-21 | A kind of alpha-1,4-dextran and its production and use | |
| PCT/CN2014/077978 WO2014187316A1 (en) | 2013-05-21 | 2014-05-21 | α-1,4-GLUCAN AND PREPARATION METHOD AND USE THEREOF |
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| CN201310190825.XA CN104177510B (en) | 2013-05-21 | A kind of alpha-1,4-dextran and its production and use |
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| CN104177510A true CN104177510A (en) | 2014-12-03 |
| CN104177510B CN104177510B (en) | 2016-11-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN113480677A (en) * | 2021-08-03 | 2021-10-08 | 青岛科技大学 | Cortex moutan linear alpha-D-1, 4-glucan and preparation method and application thereof |
| CN115990187A (en) * | 2022-12-07 | 2023-04-21 | 北京中医药大学 | Traditional Chinese medicine extract for improving Alzheimer disease and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106832042A (en) * | 2017-02-03 | 2017-06-13 | 中国科学院上海药物研究所 | The glucans of β 1,3, its preparation method and pharmaceutical applications |
| CN106832042B (en) * | 2017-02-03 | 2019-05-17 | 中国科学院上海药物研究所 | Beta-1,3-dextran, preparation method and pharmaceutical applications |
| CN113480677A (en) * | 2021-08-03 | 2021-10-08 | 青岛科技大学 | Cortex moutan linear alpha-D-1, 4-glucan and preparation method and application thereof |
| CN115990187A (en) * | 2022-12-07 | 2023-04-21 | 北京中医药大学 | Traditional Chinese medicine extract for improving Alzheimer disease and application thereof |
| CN115990187B (en) * | 2022-12-07 | 2024-02-06 | 北京中医药大学 | Traditional Chinese medicine extract for improving Alzheimer disease and application thereof |
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| WO2014187316A1 (en) | 2014-11-27 |
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