CN101235098A - Method for modifying functional plants polysaccharide - Google Patents
Method for modifying functional plants polysaccharide Download PDFInfo
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- CN101235098A CN101235098A CNA2008100590836A CN200810059083A CN101235098A CN 101235098 A CN101235098 A CN 101235098A CN A2008100590836 A CNA2008100590836 A CN A2008100590836A CN 200810059083 A CN200810059083 A CN 200810059083A CN 101235098 A CN101235098 A CN 101235098A
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- 230000004913 activation Effects 0.000 claims description 5
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- OIFBSDVPJOWBCH-UHFFFAOYSA-N Diethyl carbonate Chemical compound CCOC(=O)OCC OIFBSDVPJOWBCH-UHFFFAOYSA-N 0.000 claims description 3
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- HCXJFMDOHDNDCC-UHFFFAOYSA-N 5-$l^{1}-oxidanyl-3,4-dihydropyrrol-2-one Chemical group O=C1CCC(=O)[N]1 HCXJFMDOHDNDCC-UHFFFAOYSA-N 0.000 claims description 2
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 2
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to a modification method of functional plant polysaccharide, which comprises extracting functional polysaccharide from the plant with biological activity, separating, purifying and determining molecular weight and biological function, modifying structure via agent, activating active hydroxyl on sugar chain, connecting small molecule polyethylene imine, dialyzing, freezing and drying to obtain functional plant polysaccharide. The modification method has simple process, high efficiency and high yield, which keeps prior activity of natural polysaccharide and adds new biological function to obtain a natural macromolecule compound as non-viral gene carrier.
Description
Technical field
The present invention relates to a class natural macromolecular material, specifically relate to a class functional plants polysaccharide modifying method.
Background technology
Polysaccharide be present in natural aldose and (or) polymkeric substance that links together by glycosidic link of ketose.Polysaccharide is the requisite compositions of all lived organisms, and it has close getting in touch with all physiological functions that earn a bare living.In recent years, the polysaccharide in sources such as plant, marine organisms and mushroom is as there being important kind in the bioactive natural product to occur, and that various polysaccharide had was antitumor, immune, anticoagulation, hypoglycemic and antiviral activity are found in succession.Polysaccharide has complicated structure, and this is because it has many different monosaccharide residues, different link position and dissimilar glycosidic links, can form to have different conformations, different relative molecular masses, and the interior secondary structure with interchain hydrogen bond of chain.It should be noted that especially to have a large amount of activity hydroxies in the polysaccharide molecule structure, after special linking agent activation, can connect different compounds, thereby increase new function.
The genomic medicine treatment is the novel method of treatment that recent two decades rises, and its elementary tactics is that foreign gene is imported the purpose cell and obtains effective expression by all kinds of carriers, thereby reaches the purpose of treatment disease.In the genomic medicine delivery systme, the research of carrier is the focus of research all the time.The system of the virus vector of widespread usage mediation at present, though it has very high gene transfection efficient, but cytotoxicity, lower target, the problems such as carrying capacity, Production and Packaging and high price of restricted dna have limited the range of application of virus vector, especially 1999, U.S.'s gene therapy clinical experiment causes " JesseGelsinger " death incident because of using adenovirus carrier, makes the investigator more careful in the clinical use of viral vector.On the contrary, it is low that the non-viral transgene carrier has immune response, and the safety performance height is easy to characteristics such as synthetic, comes into one's own day by day in the research of genomic medicine.
Non-virus type genophore exists problems such as toxicity, biocompatibility, degradation property, a kind of synthetic material polymine (PEI) is arranged at present, experiment finds that the polymine transfection efficiency of molecular weight 22000-25000 is very high, but the problem that has toxicity and degradation property, the polymine toxicity of small molecular weight (600,1200,2000) is little, be easy to metabolism in vivo, but almost do not have transfection efficiency.
Kim (Kazuyoshi Sagara and Sung Wan KimJournal of Controlled Release, Volume 79, Issues 1-3,19February 2002, Pages 271-281) seminar, report the transfection efficiency that improves polymine with low molecular weight polyethylene imine beautify polyoxyethylene glycol, reduced the toxicity of polymine; Gosselin (Gosselin MA, Guo WJ, Lee RJ, BIOCONJUGATE CHEMISTRY 12 (6): 989-994 NOV-DEC 2001) etc., by cystine linkage low-molecular-weight polymine is coupled together, the polymine of the cross-linking products that obtains transfection efficiency ratio 25KD in the in-vitro transfection experiment is high 2 times, and toxicity and low molecular weight polyethylene imines are similar; Kam W.leong (Kam W.leong, etc, Bioconjug Chem.2006Jan-Feb; 17 (1): 152-8.) seminar with low-molecular-weight polymine coupling chitosan as gene drug carriers;
Yi-Yan Yang (Peggy Chan, Motoichi Kurisawa, Joo Eun Chung and Yi-Yan Yang, Biomaterials, Volume28, Issue 3, and January 2007, Pages 540-549) seminar has reported with the polyethylene coupling chitosan as gene drug carriers.
The present invention is skeleton with the functional plants polysaccharide, modifies with special connection reagent, connects low-molecular-weight polyethylene industry amine as novel non-viral gene pharmaceutical carrier.That functional plants polysaccharide after its advantage is to modify has is biodegradable, hypotoxicity and high gene transfection efficient.
Summary of the invention
The object of the invention provides the modifying method of functional plants polysaccharide, obtain functional plants polysaccharide through chemically modified, make the functional polysaccharide material both keep the original activity of natural polysaccharide, increase new biological function again, become the natural high moleculer eompound of non-viral gene vector material.The vegetable polysaccharides immunogenicity is low, is easy to biological degradation, itself has certain biologic activity.Be rich in activity hydroxy in the polysaccharide modular construction, be easy to be activated and modify.The present invention makes it become the genophore of the non-virus type of a class after selecting vegetable polysaccharides to carry out chemically modified as basic framework.
The modifying method of functional plants polysaccharide provided by the invention, total inventive concept is: by extraction functionality polysaccharide in the biologic activity plant, behind clear and definite molecular weight of separation and purification and biological function, carry out structural modification with special reagent, by the active hydroxyl on the activation glycosyl, connect small molecules polymine (PEI), the functional plants polysaccharide that obtains through the dialysis frost drying.
The modifying method of functional plants polysaccharide provided by the invention, comprise the steps: 1) the polysaccharide extraction and purification: from plant material, extract Crude polysaccharides with poach, ethanol sedimentation method, dialyse with dialysis tubing, separate with sephadex column again, separated product gel chromatography molecular weight distribution obtains the vegetable polysaccharides of the biologic activity of number-average molecular weight homogeneous within the specific limits; 2) polysaccharide structures is modified: get the polysaccharide of purifying, be dissolved in the phosphate buffered saline buffer; The linking agent of activation hydroxyl is dissolved in the methylene dichloride, under nitrogen protection, adds in the polysaccharide soln, the limit edged stirs, and adds in 90-120 minute, at room temperature reacts after adding 60-90 minute, obtains the activatory polysaccharide soln; With the small molecular weight polymine; in the phosphate buffer soln; add catalyzer; join under lucifuge, nitrogen protection, the room temperature in the activatory polysaccharide soln; the limit edged stirs; add in 120-150 minute, added under back lucifuge, the room temperature reaction 120-150 minute, the solution that reaction is finished is through dialysis, freezing, dry.Obtain the functional plants polysaccharide of the polymkeric substance of polysaccharide and small molecules polymine.
The reaction formula that carries out structural modification of the present invention is as follows:
N is the number of polysaccharide modular construction in the reaction formula, and this numerical value has directly reflected the size of polysaccharide molecular weight.
M in the reaction formula
1Reflected the unitary quantity of primary amino among the PEI, m
2Reflected the unitary quantity of secondary amino group, m
1And m
2Numerical value relevant with the molecular weight of PEI.
The vegetable polysaccharides of biologic activity of the present invention is: in lentinan, root of large-flowered skullcap polysaccharide, ganoderan, rice benevolence polysaccharide, cactus polyoses, krestin, the lycium barbarum polysaccharide any, molecular weight distribution is 10000-1100000.
The linking agent that can activate hydroxyl of the present invention is: N, N '-carbonyl dimidazoles, benzotriazole carbonic ether, chloro-formic ester, carbonylic imidazole, N, in N '-two succinimido sulfuric ester, the N-hydroxy-succinamide chloro-formic ester any.
The molecular weight of small molecular weight polymine of the present invention be in 600,1200,2000 the polymine any.
The weightmeasurement ratio of small molecular weight polymine of the present invention and phosphate buffered saline buffer is that 20-50 doubly measures.
The polysaccharide of purifying of the present invention and the weightmeasurement ratio of phosphate buffered saline buffer are that 20-50 doubly measures.
The polysaccharide amount weight ratio that linking agent consumption of the present invention is a purifying is 1-2: 3-5.
The weightmeasurement ratio of linking agent of the present invention and methylene dichloride is that 25-50 doubly measures.
Catalyzer of the present invention is a triethylamine, and consumption and small molecular weight polymine weightmeasurement ratio are 1-2: 1-5 doubly measures.
Beneficial effect of the present invention
About the polyethyleneimine: amine structure explanation of small molecular weight, the structure of polymine is as follows:
Show that from structure PEI has 3 kinds of different amine-formats; formed different buffer systems; the amino basic role that exists is in conjunction with DNA; the function of expansion be when carrying DNA and enter cell different amino to be combined into different bufferings right, can protect DNA to exempt from lysosome degraded in the tenuigenin.
Functional polysaccharide of the present invention connects micromolecular polymine after handling with linking agent.Functional plants polysaccharide has certain biologic activity, and used linking agent has special structure, and the hydroxyl on can modified polysaccharide activates latter linked small molecules polymine and keeps original 26S Proteasome Structure and Function.
The present invention is with extracted form natural plant activeconstituents-polysaccharide, on the original active basis of reservation polysaccharide, it is carried out structural modification, this modifying method advantage is simple efficient, rate of recovery height, activated hydroxyl can connect any compound with active hydrogen molecule, and therefore the expansion to the polysaccharide effect has very important significance.Connect micromolecular polymine behind the active hydroxyl on the activated glycosyl, give the natural function polysaccharide new biological function, make it have hypotoxicity, the high gene transfection efficiency, and biodegradable.Become the class natural high moleculer eompound in the non-viral gene vector material.
Modifying method of the present invention not only kept polysaccharide original activity function, more given its brand-new biological function as genophore.In addition, learn purposes for the other biological of polysaccharide and opened up new approach.
Embodiment
Below in conjunction with embodiment the present invention is carried out the description of detail, but be not limited to the disclosed content of embodiment.
Embodiment 1:
1) polysaccharide extraction and purification
Get any 500 gram in the plant medicinal plant mushroom, the root of large-flowered skullcap, glossy ganoderma, Mi Ren, Root and stem of Cholla, rainbow conk, matrimony vine of biologic activity, adding 3 times of water gagings boiled 3 hours, join in 95% ethanolic soln after the gained solution concentration, the limit edged stirs, make that final alcohol concn is 65%, after centrifugal, the gained precipitation gets Crude polysaccharides 22.5 grams through lyophilize.
Get Crude polysaccharides 10 grams, be dissolved in the 200 ml water solution, the molecular weight of packing into after the dissolving is dialysis 48 hours in 15000 the dialysis tubing, take out solution in the bag, add 2 gram decolorizing with activated carbon, filter, it is 15000 dialysis tubings dialysis 48 hours that filtrate is continued with molecular weight, solution centrifugal in the bag taking is got supernatant liquor, frost drying.Obtain 3 gram purified polysaccharides.
Get purified polysaccharide 2 grams, separate with sephadex column, gather effusive at first component, frost drying gets once more purified polysaccharide 1 gram.Through the gel chromatography analysis, molecular weight distribution is 10000-1100000.Through nucleus magnetic resonance with infraredly confirm as in lentinan, root of large-flowered skullcap polysaccharide, ganoderan, rice benevolence polysaccharide, cactus polyoses, krestin, the lycium barbarum polysaccharide any.
2) polyose modification
Get lentinan 0.5 gram of final purifying in the step 1, be dissolved in 10 ml phosphate buffers.Get N, N '-carbonyl dimidazoles 0.2 gram is dissolved in 6 milliliters of methylene dichloride, slowly is added in the polysaccharide soln under nitrogen protection, lucifuge condition, and other edged stirs, and adds in 90 minutes, adds back lucifuge reaction 60 minutes.
Get polymine 0.3 gram of molecular weight 600, be dissolved in 10 milliliters the phosphate buffer soln, add the 0.2ml triethylamine as catalyzer; join under lucifuge, the nitrogen protection in the activatory polysaccharide soln; the limit edged stirs, and adds in 120 minutes, adds back lucifuge reaction 120 minutes.
The solution that reaction the is finished molecular weight cut-off of packing into is in 15000 the dialysis tubing, to dialyse solution frost drying in the bag taking 48 hours.
Through the gel chromatography analysis, the molecular weight of reaction post polymerization thing is 10000-1150000, and through nucleus magnetic resonance and Infrared spectroscopy, the confirmatory reaction thing is the polymkeric substance of lentinan and small molecules polymine.
Embodiment 2:
Get purifying root of large-flowered skullcap polysaccharide 0.5 gram among the embodiment 1, be dissolved in 15 ml phosphate buffers.Get N, N '-two succinyl-industry amido sulfuric ester 0.2 gram is dissolved in 10 milliliters of methylene dichloride, under nitrogen protection, lucifuge condition, slowly is added in the polysaccharide soln, and the limit edged stirs, and adds in 120 minutes, adds back lucifuge reaction 90 minutes.
Get polymine 0.6 gram of molecular weight 1200, be dissolved in 15 milliliters the phosphate buffer soln, add the 0.2ml triethylamine as catalyzer; join under lucifuge, the nitrogen protection in the activatory polysaccharide soln; the limit edged stirs, and adds in 150 minutes, adds back lucifuge reaction 150 minutes.
The solution that reaction the is finished molecular weight cut-off of packing into is in 15000 the dialysis tubing, to dialyse solution frost drying in the bag taking 48 hours.
Through the gel chromatography analysis, the molecular weight of reaction post polymerization thing is 10000-1100000, and through nucleus magnetic resonance and Infrared spectroscopy, the confirmatory reaction thing is the polymkeric substance of root of large-flowered skullcap polysaccharide and small molecules polymine.
Embodiment 3:
Get purifying krestin 0.1 gram among the embodiment 1, be dissolved in 5 ml phosphate buffers.Get 0.3 gram benzotriazole carbonic ether, be dissolved in 10 milliliters of methylene dichloride, under nitrogen protection, lucifuge condition, slowly be added in the polysaccharide soln, other edged stirs, and adds in 100 minutes, adds afterreaction 80 minutes.
Get polymine 0.4 gram of molecular weight 2000, be dissolved in 10 milliliters the phosphate buffer soln, add the 0.3ml triethylamine as catalyzer; join under lucifuge, the nitrogen protection in the activatory polysaccharide soln; the limit edged stirs, and adds in 130 minutes, adds back lucifuge reaction 130 minutes.
The solution that reaction is finished is packed in 15000 dialysis tubings, dialyses solution frost drying in the bag taking 48 hours.
Through the gel chromatography analysis, the molecular weight of reaction post polymerization thing is 10000-1150000, and through nucleus magnetic resonance and Infrared spectroscopy, the confirmatory reaction thing is the polymkeric substance of krestin and small molecules polymine.
Claims (9)
1, a kind of modifying method of functional plants polysaccharide, comprise the steps: 1) the polysaccharide extraction and purification: from the plant material of biologic activity, use poach, the ethanol sedimentation method is extracted Crude polysaccharides, dialyse with dialysis tubing, separate with sephadex column again, separated product gel chromatography molecular weight distribution obtains the vegetable polysaccharides of the biologic activity of number-average molecular weight homogeneous within the specific limits; 2) polysaccharide structures is modified: get the polysaccharide of purifying, be dissolved in the phosphate buffered saline buffer; The linking agent of activation hydroxyl is dissolved in the methylene dichloride, under nitrogen protection, adds in the polysaccharide soln, the limit edged stirs, and adds in 90-120 minute, at room temperature reacts after adding 60-90 minute, obtains the activatory polysaccharide soln; The small molecular weight polymine is dissolved in the phosphate buffer soln; add triethylamine as catalyzer; join under lucifuge, nitrogen protection, the room temperature in the activatory polysaccharide soln; the limit edged stirs; add in 120-150 minute; added under back lucifuge, the room temperature reaction 120-150 minute, the solution that reaction is finished is through the functional plants polysaccharide of the polymkeric substance of dialysis, freezing, dry, acquisition polysaccharide and small molecules polymine.
2, the modifying method of functional polysaccharide according to claim 1 is characterized in that the reaction formula of described polysaccharide structures modification is as follows:
PEI is the small molecules polymine in the reaction formula, and n is the number of polysaccharide modular construction, and this numerical value has directly reflected the size of polysaccharide molecular weight, m in the reaction formula
1Reflected the unitary quantity of primary amino among the PEI, m
2Reflected the unitary quantity of secondary amino group, m
1And m
2Numerical value relevant with the molecular weight of PEI.
3, the modifying method of functional polysaccharide according to claim 1, it is characterized in that: the vegetable polysaccharides with biologic activity is: in lentinan, root of large-flowered skullcap polysaccharide, ganoderan, rice benevolence polysaccharide, cactus polyoses, krestin, the lycium barbarum polysaccharide any, molecular weight distribution is 10000-1100000.
4, the modifying method of functional polysaccharide according to claim 1, it is characterized in that: the linking agent of activation hydroxyl is: N, N '-carbonyl dimidazoles, benzotriazole carbonic ether, chloro-formic ester, carbonylic imidazole, N, in N '-two succinimido sulfuric ester, the N-hydroxy-succinamide chloro-formic ester any.
5, functional polysaccharide modifying method according to claim 1 is characterized in that: small molecular weight polymine molecular weight is: 600, in 1200,2000 the polymine any.
6, the modifying method of functional polysaccharide according to claim 1 is characterized in that: the polysaccharide of purifying and the weightmeasurement ratio of phosphate buffered saline buffer are that 20-50 doubly measures.
7, the modifying method of functional polysaccharide according to claim 1 is characterized in that: the polysaccharide amount weight ratio of linking agent consumption and purifying is 1-2: 3-5.
8, the modifying method of functional polysaccharide according to claim 1 is characterized in that: the weightmeasurement ratio of linking agent and methylene dichloride is that 20-50 doubly measures.
9, the modifying method of functional polysaccharide according to claim 1 is characterized in that: catalyzer is a triethylamine, and consumption and small molecular weight polymine weightmeasurement ratio are 1-2: 1-5 doubly measures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100590836A CN101235098A (en) | 2008-01-09 | 2008-01-09 | Method for modifying functional plants polysaccharide |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250984A (en) * | 2011-07-11 | 2011-11-23 | 宁波大学 | Method for preparing functional extracellular polysaccharide of lactic acid bacteria |
| CN104080812A (en) * | 2012-03-15 | 2014-10-01 | 方济各安吉利克化学联合股份有限公司 | Glycogen-based cationic polymers |
| CN106692981A (en) * | 2017-01-10 | 2017-05-24 | 郑州大学 | Preparation method and application of cationization lentinan |
| CN108976315A (en) * | 2018-08-05 | 2018-12-11 | 广州小众环保科技有限公司 | A kind of algin derivative and preparation method thereof for handling heavy metal-containing waste water |
| CN109072482A (en) * | 2016-05-12 | 2018-12-21 | Z生物科技有限公司 | Polyvalency glycan microarray platform |
| CN109310644A (en) * | 2016-04-14 | 2019-02-05 | 奇迹连结生物技术公司 | Anti-infective composition comprising plant glycogen nano particle |
| CN113501889A (en) * | 2021-07-06 | 2021-10-15 | 郑州大学 | Preparation method and application of pseudo-ginseng polysaccharide cationic derivative |
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- 2008-01-09 CN CNA2008100590836A patent/CN101235098A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250984A (en) * | 2011-07-11 | 2011-11-23 | 宁波大学 | Method for preparing functional extracellular polysaccharide of lactic acid bacteria |
| CN102250984B (en) * | 2011-07-11 | 2013-09-04 | 宁波大学 | Method for preparing functional extracellular polysaccharide of lactic acid bacteria |
| CN104080812A (en) * | 2012-03-15 | 2014-10-01 | 方济各安吉利克化学联合股份有限公司 | Glycogen-based cationic polymers |
| CN104080812B (en) * | 2012-03-15 | 2017-06-27 | 方济各安吉利克化学联合股份有限公司 | Glycogen base cationic polymer |
| CN109310644A (en) * | 2016-04-14 | 2019-02-05 | 奇迹连结生物技术公司 | Anti-infective composition comprising plant glycogen nano particle |
| CN109072482A (en) * | 2016-05-12 | 2018-12-21 | Z生物科技有限公司 | Polyvalency glycan microarray platform |
| US11656224B2 (en) | 2016-05-12 | 2023-05-23 | Z Biotech Llc | Multivalent glycan microarray platform |
| CN106692981A (en) * | 2017-01-10 | 2017-05-24 | 郑州大学 | Preparation method and application of cationization lentinan |
| CN108976315A (en) * | 2018-08-05 | 2018-12-11 | 广州小众环保科技有限公司 | A kind of algin derivative and preparation method thereof for handling heavy metal-containing waste water |
| CN108976315B (en) * | 2018-08-05 | 2021-01-08 | 广州小众环保科技有限公司 | Seaweed derivative for treating heavy metal-containing wastewater and preparation method thereof |
| CN113501889A (en) * | 2021-07-06 | 2021-10-15 | 郑州大学 | Preparation method and application of pseudo-ginseng polysaccharide cationic derivative |
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