CN103408674A - Acetylated ganoderma lucidum polysaccharide and preparation method thereof - Google Patents
Acetylated ganoderma lucidum polysaccharide and preparation method thereof Download PDFInfo
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- CN103408674A CN103408674A CN2013103779361A CN201310377936A CN103408674A CN 103408674 A CN103408674 A CN 103408674A CN 2013103779361 A CN2013103779361 A CN 2013103779361A CN 201310377936 A CN201310377936 A CN 201310377936A CN 103408674 A CN103408674 A CN 103408674A
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Abstract
The invention provides acetylated ganoderma lucidum polysaccharide and a preparation method thereof. The acetylated ganoderma lucidum polysaccharide is prepared by water extraction, dialysis, ion exchange chromatographic purification and acetylation modification. The acetylated ganoderma lucidum polysaccharide prepared by the method has an effect of stimulating macrophage to generate NO activity, wherein the effect of stimulating macrophage to generate NO activity is associated with substitution degree.
Description
Technical field
The present invention relates to edible fungi polysaccharide structure of modification technical field, relate to particularly a kind of acetylize ganoderan and preparation method thereof.
Background technology
Polysaccharide is the polymer be formed by connecting by glycosidic link by 10 above monose, isolating protein and the outer important biomacromolecule of another class of nucleic acid in organism, the textural factor of polysaccharide comprises the main chain character of polysaccharide, the higher structure of side chain character and polysaccharide molecule, wherein the sugar unit of polysaccharide main chain forms, the glycosidic link type has all directly determined the activity of polysaccharide, the type of polysaccharide side chain, the polymerization degree, distribution and the substitution value size that determined active polysaccharide thereof of side chain on polysaccharide chain, therefore for improving or change the biological activity of polysaccharide, molecular modification and the structure of modification of polysaccharide are significant.
Glossy ganoderma is that a kind of nutrition, health care are worth high macro-fungi, have the reputation of " celestial grass ", is the treasure in the traditional Chinese medicine and pharmacy treasure-house.The experimental results both domestic and external shows, glossy ganoderma has pharmacological action widely, and as anti-tumor activity, strengthen immunologic function, antiviral and anti-mutation, reducing blood-fat, the effect such as hypoglycemic, and toxicity is extremely low.The patent that the ganoderan acetylize is relevant is not yet arranged at present.
Summary of the invention
At first the present invention provides a kind of acetylize ganoderan, and it prepares with the following method:
Ganoderma sporophore carries out water extraction, dialysis and ion-exchange chromatography, obtains ganoderan;
Ganoderan is carried out to acetylation modification.
Specifically, a kind of acetylize ganoderan of the present invention prepares with the following method:
Ganoderma sporophore extracts with 80-120 ℃ of water; Then water extraction liquid is dialysed by dialysis tubing; Then carry out anion exchange chromatography, concentrate drying obtains ganoderan;
Ganoderan is carried out to acetylation modification by diacetyl oxide-pyridine method.
In particular, a kind of ganoderan acetyl derivatives of the present invention prepares with the following method:
By Ganoderma sporophore pulverizing, 80-120 ℃ hydro-thermal water extraction, obtain glossy ganoderma crude extract water extraction liquid;
Glossy ganoderma crude extract water extraction liquid is dialysed by dialysis tubing, and lyophilize obtains glossy ganoderma crude extract dry product;
Glossy ganoderma crude extract dry product is mixed with to the aqueous solution of 10-40mg/mL, 10000rpm, centrifugal 30min, get supernatant 10-50mL and purify with anion exchange chromatography;
The anion exchange chromatography purifying adopts the KTA chromatographic system specifically, first with the distilled water wash-out, again with the NaCl gradient elution of 0-2mol/L, flow velocity 4mL/min, fraction collection, with the phenolsulfuric acid method, detect the polysaccharide content of every pipe by pipe, merge each peak according to the elution curve of polysaccharide, the component that then concentrated, dialysis, dry ion column separate;
Take ion column chromatography obtained component; by 1:200-400(w/v) add dimethyl sulfoxide (DMSO); after ultra-sonic oscillation, room temperature is standing 30 minutes; by 1:1-10(w/v) add pyridine; according to 1:10-20(w/v) add N; the acetonitrile of N dimethyl aminopyridine; again by 1:1-15(w/v) add diacetyl oxide; be placed in 30 ℃ of reaction 0-8h; after taking-up, add the water termination reaction, and be transferred in dialysis tubing, respectively with tap water dialysis 1 day; distill water dialysis 2 days, lyophilize obtains the acetyl derivatives of ganoderan.
Wherein said dialysis tubing interception is 3000-3500Da;
Wherein said anion column chromatography, be DEAE-Sepharose Fast Flow anion column chromatography.
The present invention also provides the method for preparing above-mentioned ganoderan acetyl derivatives, and the method is:
Ganoderma sporophore carries out water extraction, dialysis and ion-exchange chromatography, obtains ganoderan;
Ganoderan is carried out to acetylation modification.
The polysaccharide acetylation modification is a kind of important modifying method, and the acetylize of polysaccharide can change directional property and the horizontal order of polysaccharide molecule, thereby changes the physical properties of polysaccharide, and the introducing of ethanoyl changes the stretching, extension of molecule.The monose that the acetylize ganoderan that the present invention makes does not change polysaccharide forms; but contents of monosaccharides changes; variation has all occurred in ganoderan structural state and the solvability after acetylation modification simultaneously; this is for improving or change the biological activity of polysaccharide; enlarge the range of application of glossy ganoderma, the utilization ratio that improves glossy ganoderma all has very important realistic meaning and learning value.
The present invention adopts the standby glossy ganoderma acetyl derivatives of diacetyl oxide-pyridine legal system to have the effect that significant stimulating expression of macrophage produces NO; and the ability of stimulating expression of macrophage generation NO activity is relevant with acetylizad substitution value, substitution value is that the 0.98 o'clock the highest generation of activity NO amount is 38.22 μ mol(concentration 25 μ g/mL).
Embodiment
The Ganoderma sporophore that the present invention uses is provided by academy of agricultural sciences, Shanghai City edible mushrooms;
The present invention uses KTA chromatographic system, filler DEAE-Sepharose Fast Flow all purchased from GE;
The present invention uses other reagent all purchased from Chemical Reagent Co., Ltd., Sinopharm Group (Shanghai)
Embodiment 1
After Ganoderma sporophore was pulverized, after according to the 1kg sporophore, adding the ratio of 15L water to soak 30 minutes, heated and boiled, extracted 2 hours in 100 ℃, filter, filter residue adds water by solid-liquid ratio 1:15 and extracts once again, merging filtrate, dialysis after filtrate is concentrated, lyophilize, obtain glossy ganoderma and slightly extract dry product.
Glossy ganoderma crude extract dry product is mixed with to the aqueous solution of 10-40mg/mL, 10000rpm, centrifugal 30min, get supernatant 10-50mL and adopt the KTA chromatographic system to carry out the anion exchange chromatography purifying.First with the distilled water wash-out, then with the NaCl gradient elution of 0-2mol/L, flow velocity 4mL/min, fraction collection, with the phenolsulfuric acid method, detect the polysaccharide content of every pipe by pipe, merge each peak according to the elution curve of polysaccharide, the component that then concentrated, dialysis, dry ion column separate.
Take ion column chromatography obtained component 10mg in eggplant-shape bottle, add the 3mL dimethyl sulfoxide (DMSO) in eggplant-shape bottle, after ultra-sonic oscillation, room temperature is standing 30 minutes; the pyridine that adds 50 μ L, 150 μ LN, the acetonitrile of N dimethyl aminopyridine; the diacetyl oxide that adds again 150 μ L; be placed in 30 ℃ of reaction 2h, after taking-up, add 5mL water termination reaction, and be transferred in dialysis tubing; with tap water dialysis one day; distill water dialysis 2 days, the acetylated polysaccharide acetyl content is measured in lyophilize, calculates substitution value.
Experimental result:
Acetyl content is 8.91%, and substitution value is 0.37.
Wherein the acetyl content measuring method is oxammonium hydrochloride-iron trichloride-salt acid system
The ethanoyl calculation formula is:
Embodiment 2
Take the ion column chromatography obtained component 10mg of preparation in embodiment 1 in eggplant-shape bottle; in eggplant-shape bottle, add the 3mL dimethyl sulfoxide (DMSO); after ultra-sonic oscillation, room temperature is standing 30 minutes; the pyridine that adds 50 μ L; 150 μ LN; the acetonitrile of N dimethyl aminopyridine; the diacetyl oxide that adds again 150 μ L; be placed in 30 ℃ of reaction 4h, after taking-up, add 5mL water termination reaction, and be transferred in dialysis tubing; with tap water dialysis one day; distill water dialysis 2 days, the acetylated polysaccharide acetyl content is measured in lyophilize, calculates substitution value.
Experimental result:
Acetyl content is 20.07%, and substitution value is 0.98.
Embodiment 3
Take the ion column chromatography obtained component 10mg of preparation in embodiment 1 in eggplant-shape bottle, add the 3mL dimethyl sulfoxide (DMSO) in eggplant-shape bottle, after ultra-sonic oscillation, room temperature is standing 30 minutes; the pyridine that adds 50 μ L; 150 μ LN, the acetonitrile of N dimethyl aminopyridine, then add the diacetyl oxide of 150 μ L; add at once 5mL water termination reaction; and be transferred in dialysis tubing, with tap water dialysis one day, distill water dialysis 2 days; the acetylated polysaccharide acetyl content is measured in lyophilize, calculates substitution value.
Experimental result:
Acetyl content is 1.98%, and substitution value is 0.08.
Embodiment 4
Take the ion column chromatography obtained component 10mg of preparation in embodiment 1 in eggplant-shape bottle; in eggplant-shape bottle, add the 3mL dimethyl sulfoxide (DMSO); after ultra-sonic oscillation, room temperature is standing 30 minutes; the pyridine that adds 50 μ L; 150 μ LN; the acetonitrile of N dimethyl aminopyridine; the diacetyl oxide that adds again 30 μ L; be placed in 30 ℃ of reaction 4h, after taking-up, add 5mL water termination reaction, and be transferred in dialysis tubing; with tap water dialysis one day; distill water dialysis 2 days, the acetylated polysaccharide acetyl content is measured in lyophilize, calculates substitution value.
Experimental result:
Acetyl content is 11.09%, and substitution value is 0.47.
Embodiment 5
Take the ion column chromatography obtained component 10mg of preparation in embodiment 1 in eggplant-shape bottle; in eggplant-shape bottle, add the 3mL dimethyl sulfoxide (DMSO); after ultra-sonic oscillation, room temperature is standing 30 minutes; the pyridine that adds 50 μ L; 150 μ LN; the acetonitrile of N dimethyl aminopyridine; the diacetyl oxide that adds again 50 μ L; be placed in 30 ℃ of reaction 4h, after taking-up, add 5mL water termination reaction, and be transferred in dialysis tubing; with tap water dialysis one day; distill water dialysis 2 days, the acetylated polysaccharide acetyl content is measured in lyophilize, calculates substitution value.
Experimental result:
Acetyl content is 17.63%, and substitution value is 0.80.
Embodiment 6 stimulated in vitro scavenger cells generate the NO experiment
The strain of RAW264.7 bone marrow macrophage, purchased from (the ATCC number TIB-71 of American National DSMZ
TM);
DMEM, RPMI1640 are purchased from GIBCO company.
The preparation of test sample
Accurately take acetylize ganoderan sample prepared by embodiment 1-5 in sterilizing eppendorf pipe, be configured to concentration 5mg/mL with the PBS damping fluid.After fully dissolving, with the centrifugal 30min of 12000 rpm/min, under aseptic condition, be transferred in new sterile eppendorf tubes, each sample be diluted to respectively to 10,50,250 μ g/mL stand-by.Negative control is the PBS damping fluid, and positive control is 10 μ g/mL LPS solution.
The preparation of mouse RAW264.7 scavenger cell
With the DMEM substratum at 37 ℃, 5% CO
2Under condition, go down to posterity after cultivation, use 0.25% tryptic digestion, collecting cell after the centrifugal 3min of suspension 300 * g, with colourless RPMI1640 substratum, that cell dilution is standby to finite concentration.
Scavenger cell discharges the mensuration of NO activity
Because NO is extremely unstable, generate very soon in vivo oxynitroso (NO2
-) and nitroxyl (NO3
-), therefore adopt the NO2 in Griess method working sample
-/ NO3
-As the index of weighing the NO level.
(1) with Sodium Nitrite typical curve processed:
Be made into the sodium nitrite solution of different concns, concentration gradient is 0,5,10,15,20,25,30,35,40 μ M totally 9 concentration gradients; Get 100 μ L in 96 orifice plates, add 50 μ L Griess reagent, measure the 543nm light absorption value, 3 repetitions of each concentration of typical curve, drawing standard curve.
(2) NO determination of yield
After the digestion of the nutrient solution of scavenger cell with colourless RPMI1640 substratum (containing 10% foetal calf serum, 1% antibiotic liquid) by cell dilution to 5 * 10
5/ ml, every hole 180 μ L add 96 orifice plates, and then add each concentration testing sample of 20 μ L and feminine gender (PBS), positive (LPS, 10 μ g/mL) contrast, in 37 ℃, contain under 5% CO2 condition and cultivate 48h after, get 100 μ L culture supernatant in 96 orifice plates, add 50 μ L Griess reagent, after colour developing 10min, in wavelength 543nm place, measure absorbancy, calculate corresponding NO amount according to typical curve.
Experimental result:
Substitution value be 0.08,0.37,0.47,0.8,0.98 acetylize ganoderan when final concentration 1 μ g/mL, stimulating expression of macrophage produces the amount (unit: μ moL/5.0 * 10 of NO
5Cells) be respectively: 7.33,14.56,18.30,14.64,15.78; When final concentration 5 μ g/mL, produce the NO amount and be respectively 8.87,22.93,25.05,21.23,30.17; When final concentration 25 μ g/mL, produce the NO amount and be respectively 22.46,26.67,31.15,37.49,38.22, negative control is 5.29, positive control (concentration 1 μ g/mL) is 49.20, sample sets shows good stimulating expression of macrophage and produces the NO activity, and stimulating expression of macrophage generation NO is active relevant with the sample substitution value.
Claims (4)
1. acetylize ganoderan it is characterized in that preparing with the following method:
Ganoderma sporophore carries out water extraction, dialysis and ion-exchange chromatography, obtains ganoderan;
Ganoderan is carried out to acetylation modification.
2. acetylize ganoderan according to claim 1 is characterized in that it prepares with the following method:
Ganoderma sporophore extracts with 80-120 ℃ of water; Then water extraction liquid is dialysed by dialysis tubing; Then carry out anion exchange chromatography, concentrate drying obtains ganoderan;
Ganoderan is carried out to acetylation modification by diacetyl oxide-pyridine method.
3. acetylize ganoderan according to claim 2, is characterized in that
Wherein said dialysis tubing interception is 3000-3500Da;
Wherein said anion column chromatography, be DEAE-Sepharose Fast Flow anion column chromatography.
4. method for preparing the described acetylize ganoderan of claim 1-3 any one is characterized in that the method is:
Ganoderma sporophore carries out water extraction, dialysis and ion-exchange chromatography, obtains ganoderan;
Ganoderan is carried out to acetylation modification.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601759A (en) * | 2016-01-07 | 2016-05-25 | 广西医科大学 | Acetylated longan pulp polysaccharide and application of acetylated longan pulp polysaccharide to medicine preparing |
| CN108203472A (en) * | 2017-05-03 | 2018-06-26 | 南昌大学 | A kind of new antitumoral compounds obtained by acetylation |
| CN112625144A (en) * | 2020-12-30 | 2021-04-09 | 江南大学 | Acetylated soluble soybean polysaccharide and application thereof in improving milk tea stability |
| CN116035204A (en) * | 2022-12-20 | 2023-05-02 | 大连康芝源生物科技有限公司 | Edible fungus extract fermentation type energy supplementing food and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070104729A1 (en) * | 2001-08-06 | 2007-05-10 | Academia Sinica | Immuno-modulating antitumor activities of ganoderma lucidum (reishi) polysaccharides |
| CN101857645A (en) * | 2010-05-20 | 2010-10-13 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN102617745A (en) * | 2012-03-05 | 2012-08-01 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Ganoderma lucidum karst polysaccharide F31 |
-
2013
- 2013-08-27 CN CN2013103779361A patent/CN103408674A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070104729A1 (en) * | 2001-08-06 | 2007-05-10 | Academia Sinica | Immuno-modulating antitumor activities of ganoderma lucidum (reishi) polysaccharides |
| CN101857645A (en) * | 2010-05-20 | 2010-10-13 | 无限极（中国）有限公司 | Water-soluble neutral Ganoderma lucidum polysaccharide GLP1 and preparation method thereof |
| CN102617745A (en) * | 2012-03-05 | 2012-08-01 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Ganoderma lucidum karst polysaccharide F31 |
Non-Patent Citations (2)
| Title |
|---|
| 冯娜等: ""不同生育期灵芝子实体水提物与其免疫活性"", 《食用菌学报》 * |
| 叶立斌: ""灵芝子实体多糖（缀合物）的纯化、结构鉴定、分子改性和生物活性研究"", 《中国优秀博士学位论文全文数据库（电子期刊） 农业科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601759A (en) * | 2016-01-07 | 2016-05-25 | 广西医科大学 | Acetylated longan pulp polysaccharide and application of acetylated longan pulp polysaccharide to medicine preparing |
| CN105601759B (en) * | 2016-01-07 | 2018-01-19 | 广西医科大学 | A kind of acetylation Arillus longan polysaccharide and its application in pharmacy |
| CN108203472A (en) * | 2017-05-03 | 2018-06-26 | 南昌大学 | A kind of new antitumoral compounds obtained by acetylation |
| CN112625144A (en) * | 2020-12-30 | 2021-04-09 | 江南大学 | Acetylated soluble soybean polysaccharide and application thereof in improving milk tea stability |
| CN112625144B (en) * | 2020-12-30 | 2022-03-11 | 江南大学 | Acetylated soluble soybean polysaccharide and application thereof in improving milk tea stability |
| CN116035204A (en) * | 2022-12-20 | 2023-05-02 | 大连康芝源生物科技有限公司 | Edible fungus extract fermentation type energy supplementing food and preparation method thereof |
| CN116035204B (en) * | 2022-12-20 | 2024-01-02 | 大连康芝源生物科技有限公司 | Edible fungus extract fermentation type energy supplementing food and preparation method thereof |
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Application publication date: 20131127 |