CN1080726C - Process for extracting single polysaccharide from crude polysaccharideo f morel and products thereof - Google Patents
Process for extracting single polysaccharide from crude polysaccharideo f morel and products thereof Download PDFInfo
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- CN1080726C CN1080726C CN99109802A CN99109802A CN1080726C CN 1080726 C CN1080726 C CN 1080726C CN 99109802 A CN99109802 A CN 99109802A CN 99109802 A CN99109802 A CN 99109802A CN 1080726 C CN1080726 C CN 1080726C
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Abstract
The present invention relates to single polysaccharide extracted from crude polysaccharide of morel, the single polysaccharide has the biological activity, the single polysaccharide is formed by condensation of xylose, glucose, arabinose and galactoside, the single polysaccharide has beta-pyranoside keys, and the molecular weight of the single polysaccharide is 11500. Crude polysaccharide whose molecular weight is from 10 to 100 thousand, which is obtained from nutrient solution of morel, passes through a DE-52 cellulose ion exchange column and a SepharoseCL-6B gel column, the crude polysaccharide is eluted with deionized water, and through purification and separation, the single polysaccharide is obtained. The technical method is simple, the operation is convenient, and the single polysaccharide which has certain molecular weight and a determinate functional group structure can be obtained.
Description
The present invention relates to a kind of ion-exchange chromatography and gel filtration chromatography method utilized, from morel, extract the certain molecular weight of biologically active, method of the holosaccharide that structure of functional groups is determined and products thereof.
Morel is described as the famousst and precious in the world edible mushrooms at present, belongs to the large-scale food medicine of low temperature modification dual-purpose bacterium, just is put in " dish portion " in China's " wide bacterium spectrum " and Compendium of Material Medica, is called sheep tripe dish.Its meat is tender and crisp, and is fragrant and sweet good to eat, is one of treasure in the edible mushrooms.Modern medicine clinical showing contained anticancer effective ingredient in the morel, have strengthening immunity, and be antitumor, health care wait for a long time the aspect effect and more and more paid attention to by common people.Edible fungi polysaccharide has the unique biological characteristic and is subjected to the people parent day by day and looks at, as: lentinan, Chinese scholartree fungus polysaccharides etc. are used to raise immunity, treatment hepatitis and tumour.Because the complicacy of polysaccharide and all restrictions of separation and analytical procedure in the morel, the kind of Morchella esculenta (L.) Pers polysaccharide is not verified so far as yet, and its structure is not definite fully yet, and the report that the structure and its pharmacological action of Morchella esculenta (L.) Pers polysaccharide concerned also seldom.People are to the understanding of morel and use that also to rest on genuine dosage big, carry and eat all inconvenient nutrition stock solution stage, at present, the separation of morel is purified, taking effective bioactive mixed polysaccharide, is a problem of modern medicine and pharmacology research.
Purpose of the present invention is utilized methods such as ion-exchange chromatography method and gel filtration chromatography exactly, extracts the certain molecular weight of biologically active from the morel nutritive medium, the holosaccharide that structure of functional groups is determined.
Technical scheme of the present invention is such: the employing deionized water is an elutriant, by cellulose ion exchange column and gel column, will be by wood sugar, glucose, pectinose, semi-lactosi, mol ratio is 0.29: 0.24: 0.61: 0.39 condensation forms, and has β-pyranose glycosidic bond, molecular weight and be molecular weight that 11500 mixed polysaccharide obtains and be in the Crude polysaccharides of 1-10 ten thousand to separate from the morel nutritive medium.At first load the ion-exchange cellulose post, get a certain amount of DEAE, call the DE-52 Mierocrystalline cellulose in the following text, it is immersed in the damping fluid that analytically pure three (methylol) aminomethanes and hydrochloric acid is mixed, making it the pH value is 9.6, makes ion-exchange cellulose become the OH-type.The DE-52 Mierocrystalline cellulose that soaked is inserted in the glass column, with the damping fluid that peristaltic pump input three (methylol) aminomethanes and hydrochloric acid are mixed, pH9.6, balance reasonable time.The second step molecular weight that will extract from the morel nutrient solution with hyperfiltration process is that the Crude polysaccharides of 1-10 ten thousand is dissolved in the deionized water, and the deionized water consumption is as the criterion for abundant dissolving Crude polysaccharides, and lysate is directly slowly gone up in column cap.With peristaltic pump deionized water is injected the ion-exchange cellulose post, flow velocity is 0.8-1.2ml/min, and the time is 6-10 hour, and temperature is a room temperature.Collect a pipe with run tank with every 5min, elutriant is collected, do phenol one sulfuric acid process with the UV250PC ultraviolet-visible pectrophotometer then and detect the polysaccharide peak position, merge the simple spike polysaccharide, have the elutriant on maximum absorption peak to collect being determined at 490nm.After the lyophilize, this single polysaccharide is dissolved in the distilled water, the distilled water consumption is as the criterion with this single polysaccharide of abundant dissolving, then with its upper prop in the sepharose column cap, call Sepharose CL-6B gel column cap in the following text, with distilled water thorough washing, collection, to collect liquid and detect the polysaccharide peak position through phenol one sulfuric acid process again, be collected in the high collection liquid that absorbs peak position of 490nm, after the desalination lyophilize, obtain the cotton-shaped single polysaccharide finished product of white cotton.
Single polysaccharide is done following mensuration:
Single polysaccharide is measured infrared spectra routinely through the KBr compressing tablet, is H-NMR subsequently and reaches
13The C-NMR spectral analysis of the nuclear magnetic resonance.
1. molecular weight and purity testing: this experiment is identified purity of polysaccharide with high performance liquid chromatography, and measures its molecular weight.High performance liquid chromatograph is Waters 600, Ultrahydrogel 2000 and the series connection of Ultrahydrogel 500 chromatographic columns, Waters 410 type differential refraction detectors.Moving phase is the 0.1mol/l phosphate buffered saline buffer, and flow velocity is 0.6ml/min.
Recording the single polysaccharide molecular weight is 11500.
Record single polysaccharide and on Waters HPLC, be single symmetrical peak.
2. high performance capillary electrophoresis analysis
Get single polysaccharide 10mg and be dissolved in the 1ml 1mol/L sulfuric acid, tube sealing hydrolysis 10h, neutralization, centrifugal, carry out HPCE after supernatant liquor and alpha-naphthylamine are derived and analyze.After deriving with quadrat method, mixed monose standard employing analyzes.HPCE experiment condition: voltage 18KV, electric current 46 μ A, ultraviolet detection wavelength 254nm, borate buffer solution 100mmol/l, pH10.0.0.39) etc. (mol ratio is 0.29: 0.24: 0.61: four kinds of monosaccharide residues are the mixed polysaccharide that repeating unit is formed to this single polysaccharide by wood sugar, glucose, pectinose, semi-lactosi after measured.
3. UV spectrum is not seen the charateristic avsorption band of protein and nucleic acid at 260nm and 280nm place.Results of elemental analyses is a carbon containing 39.11%, and hydrogeneous is 8.98%, nonnitrogenous.
4. infrared spectra is presented at 4000cm
-1-650cm
-1The general feature that has polysaccharose substance in the district.At 3500cm
-1Absorption peak be the stretching vibration peak of free OH, at 1024cm
-1And 1039cm
-1Ehter bond C-O-C vibration absorption peak for pyranose ring; At 890cm
-1Near the charateristic avsorption band of β-glycosidic link is arranged, and at 840cm
-1The place does not have the charateristic avsorption band of α-glycosidic link.
1The resonance signal of δ 4.80ppm, i.e. C among the H-NMR
1The fignal center in proton district proves that further glycosidic link is the β type below 5.0ppm.
Experiment showed, that more than the single polysaccharide that the present invention obtains is that (mol ratio is 0.29: 0.24: 0.61: 0.39) condensation forms, and the molecular weight with β-pyranose glycosidic bond is 11500 mixed polysaccharide by wood sugar, glucose, pectinose, semi-lactosi.
With exchange of DE-52 cellulose ion and Sepharose CL-6B, with the deionized water is elutriant, the morel raw sugar is carried out purifies and separates obtain four kinds of monose (wood sugar, glucose, pectinose, semi-lactosi) residue forms for the repeating unit condensation, its molecular weight is 11500, mixed polysaccharide with β-pyranose glycosidic bond, technology is simple, easy to operate, can obtain the certain molecular weight of biologically active, the holosaccharide that structure of functional groups is determined is for next step research Morchella esculenta (L.) Pers polysaccharide biological activity with move towards higher level biochemical market and lay the first stone.
Fig. 1 Morchella esculenta (L.) Pers polysaccharide elution curve on DE-52.
Fig. 2 single polysaccharide gel permeation chromatography elution curve.
Fig. 3 high performance liquid chromatography is to the molecular weight determination figure of single polysaccharide.
The HPCE figure of Fig. 4 standard monose and single polysaccharide hydrolyzate.
Among the figure: 1. wood sugar, 2,4. seminose, 3. glucose, 5. pectinose, 6. semi-lactosi
The infrared spectrum of Fig. 5 single polysaccharide.
At first prepare the DE-52 cellulose column, get enough DE-52 Mierocrystalline celluloses, it is immersed in analytically pure three (methylol) aminomethane buffer solution, its pH value is transferred to 9.6, make the DE-52 Mierocrystalline cellulose become OH with HCl
-Type.Then soaked DE-52 Mierocrystalline cellulose is packed in the glass column of Φ a 2.6 * 90cm, with peristaltic pump input three (methylol) aminomethane, the filler balance is two days in the buffered soln coupled columns of pH9.6.From the morel nutrient solution, extract the Crude polysaccharides 100mg that molecular weight is 1-10 ten thousand by ultrafiltration, be dissolved in the deionized water of 1ml and dissolve, it directly is splined on DE-52 Mierocrystalline cellulose glass column cap, with peristaltic pump deionized water is injected the DE-52 cellulose column, flow velocity is 0.93ml/min, wash-out Crude polysaccharides 9 hours, with run tank elutriant being collected a pipe with per 5 minutes collects, detect the polysaccharide peak position of collecting liquid with the phenolsulfuric acid method, instrument is day island proper Tianjin UV2501PC ultraviolet spectrophotometer, is collected in the elutriant that 490nm has the maximum absorption peak, after its lyophilize, obtain white powder, this single polysaccharide white powder is dissolved in the 1ml distilled water again, it is splined on Sepharose CL-6B gel column cap, (gel column is Φ 1.6 * 80cm), cross post, collect.With the polysaccharide peak position of phenolsulfuric acid method detection elutriant, collect the elutriant of high absorption value peak position, lyophilize obtains the cotton-shaped single polysaccharide 30.1mg of a kind of white cotton.Through the KBR compressing tablet, measure infrared spectra routinely, be H-NMR subsequently and reach
13The C-NMR spectral analysis of the nuclear magnetic resonance, the cotton-shaped single polysaccharide of this white cotton is wood sugar, glucose, pectinose, the molecular weight with β-pyranose glycosidic bond that the semi-lactosi condensation forms are 11500 mixed polysaccharide.
Embodiment 2
Method according to embodiment 1 obtains 1g morel Crude polysaccharides, and it is dissolved in the deionized water of 8ml, makes the DE-52 cellulose column of Φ 2.6 * 90cm with the method for embodiment 1, with the dosing upper prop, use the deionized water wash-out, flow velocity is 1.15ml/min, and the time is 8 hours.Collect elutriant with method similarly to Example 1, obtain white powder after the lyophilize, white powder is dissolved in the distilled water,, obtain the byssaceous single polysaccharide of 301mg at last through the processing of Sepharose CL-6B gel column.Through the KBr compressing tablet, measure infrared spectra routinely, do spectral analysis of the nuclear magnetic resonance, this flocculence single polysaccharide is wood sugar, glucose, pectinose, the molecular weight with β-pyranose glycosidic bond that the semi-lactosi condensation forms are 11500 mixed polysaccharide.
Extracting the solid molecular weight with β-pyranose glycosidic bond that forms that closes of wood sugar, glucose, pectinose, semi-lactosi with embodiment 1 same processing method from the morel Crude polysaccharides is 11500 mixed polysaccharide.Get morel Crude polysaccharides 1000g, the DE-52 cellulose column is Φ 0.5 * 2m, and the deionized water flow velocity is 0.8ml/min, and the time is 10 hours, and Sepharese CL-6B gel column suitably increases volume in proportion.Obtain the cotton-shaped single polysaccharide of 301g white cotton at last.Through the KBr compressing tablet, carry out infrared spectra routinely, do spectral analysis of the nuclear magnetic resonance subsequently, the cotton-shaped single polysaccharide of this white cotton is the preextraction thing.
Claims (8)
1. single polysaccharide that from the morel Crude polysaccharides, extracts, it is characterized in that: it is a kind of by wood sugar, glucose, pectinose, semi-lactosi, mol ratio is 0.29: 0.24: 0.61: 0.39 condensation forms, and has β-pyranose glycosidic bond, and its molecular weight is 11500 mixed polysaccharide.
2. the extracting method of the described a kind of single polysaccharide that from the morel Crude polysaccharides, extracts of claim 1, it is characterized in that: adopting with the deionized water is elutriant, by cellulose ion exchange column and gel column, wood sugar, glucose, pectinose, semi-lactosi condensation are formed, have β-pyranose glycosidic bond, its molecular weight is that molecule that 11500 mixed polysaccharide obtains from the morel nutritive medium is to separate in the Crude polysaccharides of 1-10 ten thousand.
3. the extracting method of the single polysaccharide that extracts in a kind of morel Crude polysaccharides according to claim 2, it is characterized in that: the fill of cellulose ion exchange column is a DEAE.
4. the extracting method of a kind of single polysaccharide that extracts from the morel Crude polysaccharides according to claim 3 is characterized in that: DEAE is to convert OH to after soaking through the damping fluid of three (methylol) aminomethanes and hydrochloric acid
-The Mierocrystalline cellulose of type.
5. the extracting method of a kind of single polysaccharide that extracts from the morel Crude polysaccharides according to claim 2 is characterized in that: the deionized water elution time is 8-10 hour, and the flow velocity of water is the 0.8-1.2 ml/min, and temperature is a room temperature.
6. the extracting method of a kind of single polysaccharide that extracts from the morel Crude polysaccharides according to claim 2 is characterized in that: the weighting material of gel column is a sepharose.
7. the extracting method of a kind of single polysaccharide that from the morel Crude polysaccharides, extracts according to claim 3, it is characterized in that: when the fill of cellulose ion exchange column is DEAE, to elutriant by the DEAE ion exchange column, detect its polysaccharide peak position with the phenolsulfuric acid method, be collected in the elutriant lyophilize that 490nm has the maximum absorption peak.
8. the extracting method of a kind of single polysaccharide that from the morel Crude polysaccharides, extracts according to claim 6, it is characterized in that: when the weighting material of gel column is sepharose, elutriant by the sepharose post detects its polysaccharide peak position with the phenolsulfuric acid method, is collected in the elutriant lyophilize that 490nm has the maximum absorption peak.
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| CN1080726C true CN1080726C (en) | 2002-03-13 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103130909A (en) * | 2013-03-17 | 2013-06-05 | 吉林大学 | Preparation method of selenium-rich Morchella polysaccharide |
| CN103969384B (en) * | 2014-05-06 | 2016-04-20 | 济南康众医药科技开发有限公司 | A kind of content assaying method of blood clam polysaccharide |
| CN108828103B (en) * | 2018-08-21 | 2021-02-19 | 辽宁省农业科学院 | Morchella HPCE fingerprint establishing method and standard fingerprint thereof |
| CN113150181B (en) * | 2021-05-06 | 2022-07-15 | 四川省食用菌研究所 | Morchella extract and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4062941A (en) * | 1975-06-11 | 1977-12-13 | G. D. Searle & Co. Ltd. | Method for treating fungal infections using cell lytic enzymes |
| JPH0352879A (en) * | 1989-07-19 | 1991-03-07 | Taisho Pharmaceut Co Ltd | Intermediate for muscarine |
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1999
- 1999-07-14 CN CN99109802A patent/CN1080726C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4062941A (en) * | 1975-06-11 | 1977-12-13 | G. D. Searle & Co. Ltd. | Method for treating fungal infections using cell lytic enzymes |
| JPH0352879A (en) * | 1989-07-19 | 1991-03-07 | Taisho Pharmaceut Co Ltd | Intermediate for muscarine |
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