CN1648136A - Companumoea root polyose, derivative and its preparing method and use - Google Patents
Companumoea root polyose, derivative and its preparing method and use Download PDFInfo
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Abstract
Companumoea polyose is obtained through extracting coarse polyose from companumoea root and chromatographic purification and refining. Bioactivity test shows that companumoea polyose has obvious effects of promoting nerve cell growth and differentiation and repairing damaged nerve cell similar to nerve growth factor. Therefore, companumoea polyose may be used in medicine for preventing and treating senile dementia, learning memory disorder, children's hypophrenosis, apoplexy and other diseases, and health food for improving memory and protecting brain.
Description
Technical field: the present invention relates to a kind of Companumoea root polyose, derivative and its production and application; The technical field that belongs to medicine and protective foods.
Background technology: the have macromolecular substance of regulating body's immunity of polysaccharide for generally acknowledging, its effect shows as multipath, too many levels, many target spots property.Studies show that active polysaccharide is not to act on immunity system separately, have the balance of adjusting nerve-endocrine-immunomodulatory network (NIM).The complete regulation loop that between nerve, internal secretion and immunity system, has a various neurotransmitters, hormone and immunologic active material to constitute, the three influences each other, keep the constant of internal milieu jointly, wherein immunity system then is this integrally-regulated important component part.
Chinese medicine active polysaccharide (Polysacharides) all has wide influence to body specific immunity and non-specific immunity, cellular immunization and humoral immunization, and promotion and the effect of recovery human body immune function are extremely obvious.Polysaccharide is as biological response modifiers (BRM), main reticuloendothelial system (RES) to body, scavenger cell (M Φ), lymphocyte, white corpuscle, the NK cell, complement system and RNA, DNA, proteinic synthetic and intravital cAMP, the content of cGMP just exerts an influence, generation that finally can enhancing antibody, make the enhancing that induces of lymphokine and Interferon, rabbit, thereby reach antibiotic, antiviral, parasiticide, antitumor, radioprotective, anti-ageing action effect (the Nie Wei that waits for a long time, Zhang Yongxiang, immuno-modulating effect of polysaccharides and study on mechanism progress thereof, the Chinese Pharmacological circular, 1999,15 (6): 484~7; Li Jie, Zhang Chujing, the immunomodulatory of herbal polysaccharide and antitumor research overview in recent years, Tianjin pharmacy, 2003,15 (6): 62-4).
The natural polysaccharide that has been separated to has kind more than 100, but is applied to clinical to have only severally, to the exploitation of the little polysaccharide of steady quality, determined curative effect, toxicity and untoward reaction, particularly uses as medicine, and is significant.According to carbohydrate biological Characteristics Study result, have the people once to foretell, polysaccharide medicine might to the treatment senile dementia effectively (Thomas.WR, CurrentOpinioninBiotechnology, 1998,9:74-79).Nerve growth factor is the medicine that is hopeful to develop into anti-senile dementia disease most, seeks the polysaccharide with nerve growth factor effect from Chinese medicine, has the potentiality that develop into prevention and treatment senile dementia disease drug.
The Chinese medicine active polysaccharide to the effective research report of neurocyte seldom, existing patent report Oleander flower polysaccharide has nerve growth factor effect (Chinese patent, application number: 99125746, publication number CN 1301774, denomination of invention: Folium seu Cortex Nerii polysaccharide and derivative thereof, preparation method and application thereof); In addition, the Root of Upright Ladybell polysaccharide can improve learning and memory in rats obstacle due to the Scopolamine (Zhang Chunmei, Li Xinfang, Root of Upright Ladybell polysaccharide can improve the research of learning and memory in rats obstacle due to the Scopolamine, Chinese pharmacology and clinical, 1002,17 (6): 19-21).
Summary of the invention: we have a neurotrophic effect in carrying out ethnic drug material research the time filters out the Guizhou Province and produces dietotherapeutic plant Root of Java Campanumoea (leopard platymiscium leopard from numerous plants, Campanumoeajavanica Bl.) polysaccharide has the nerve growth factor effect, promptly neurocyte is had promotes growth and Differentiation, can repair the function that neurocyte decreases.
For this reason, the present invention is in order to reach following purpose:
1) provides a kind of Companumoea root polyose and derivative thereof with nerve growth factor effect.
2) provide the preparation method and the approach of this Companumoea root polyose, it is by extraction in the piece root of Root of Java Campanumoea, separation, purifying and get.
3) provide the preparation method of the derivative of Companumoea root polyose: Companumoea root polyose is through sulfation and carboxymethylation and get.
4) provide the application of Companumoea root polyose in medicine: prevention and treatment senile dementia, apoplexy, children mental retardation, memory dysfunction and disease such as go down.
5) provide the application of Companumoea root polyose in protective foods: improve memory dysfunction, promote or the raising learning and memory, as cerebral protective agent.
The present invention constitutes like this: the preparation method of Companumoea root polyose and derivative: from dietotherapeutic plant Root of Java Campanumoea, extract, separate and the refining Crude polysaccharides that obtains, and CPJ-1 and two kinds of holosaccharide materials of CPJ-2 of forming it.Concrete preparation method is, piece root extraction separation Companumoea root polyose by Root of Java Campanumoea, its step is as follows: the extraction of a. Crude polysaccharides preparation: with the piece root of Root of Java Campanumoea with reflow of alcohol removal of impurities, petroleum ether degreasing after, carry through the distillation hydro-thermal, extract can get the Root of Java Campanumoea Crude polysaccharides behind the alcohol precipitation after removing Deproteinization, decolouring again.B. the purifying of polysaccharide is with refining: will extract the Crude polysaccharides for preparing gets Companumoea root polyose behind DEAE cellulose chromatography and SepharoseCL-6B and Sephadex G-25 gel filtration chromatography pure substance CPJ-1 and CPJ-2 among the step a.Among the present invention, the Companumoea root polyose that obtains is carried out sulfonated and carboxymethylation, thereby obtain the derivative of Companumoea root polyose.
Described Companumoea root polyose and derivative thereof have tangible promotes growth, Differentiation and can obviously repair the injured nerve cells function neurocyte.
The application of described Companumoea root polyose and derivative thereof: Companumoea root polyose and derivative thereof are prepared into suitable form of administration, are applied in the medicine of diseases such as preparation prevention and treatment nerve degenerative diseases such as senile dementia, learning memory disorder, children mental retardation, apoplexy.
The application of described Companumoea root polyose and derivative thereof can also be: Companumoea root polyose and derivative thereof are applied in preparation improve memory dysfunction, promote or improve learning and memory, in the protective foods as cerebral protective agent.
Among the present invention: (1) has the Companumoea root polyose and the derivative thereof of nerve growth factor effect, comprises Root of Java Campanumoea Crude polysaccharides, holosaccharide component and sulfation thereof and carboxymethylation thing.
(2) preparation method of Companumoea root polyose and approach.After extracting, remove albumen, decolouring, the piece root of Root of Java Campanumoea (campanulaceae leopard platymiscium leopard Campanumoeajavanica Bl.) can get the Root of Java Campanumoea Crude polysaccharides behind the alcohol precipitation again, again to the holosaccharide component of Crude polysaccharides through separation, DEAE cellulose chromatography and SepharoseCL-6B and Sephadex G-25 gel filtration chromatography separation and purification gram.
(3) Companumoea root polyose can be got its corresponding derivative after sulfation and carboxymethylation.
(4) Companumoea root polyose and derivative thereof are applied in the medicine of diseases such as preventing and treat senile dementia, apoplexy, children mental retardation, memory dysfunction and go down.Its mode of taking can be oral, intravenous injection, transfusion etc., can be suitable form of administration such as tablet, capsule, powder pin, injection liquid, infusion solutions, wherein also can use auxilliary agent and the vehicle of adding of inertia.Other application methodes can be existing ordinary methods.
(5) Companumoea root polyose and derivative thereof the application in protective foods: improve memory dysfunction, promote or the raising learning and memory, as cerebral protective agent.Its mode of taking can be a method oral or that other are existing.
In order to reach better effect, the present invention is preferably as follows technical scheme:
One, the preparation method of Companumoea root polyose
Companumoea root polyose derives from the piece root of Guizhou Province pan money Panthera plant leopard (also can be other place of production), and its preparation process is as follows:
1, extract and separate: with the piece root of Root of Java Campanumoea with reflow of alcohol removal of impurities, petroleum ether degreasing after, carry through the distillation hydro-thermal, filter, concentrate, remove albumen, decolour after again through alcohol precipitation, can get Root of Java Campanumoea and go out polysaccharide (seeing Fig. 1 for details).
2, the purifying of polysaccharide and refining: carry out diethylaminoethyl cellulose (DEAE) column chromatography with extracting the Crude polysaccharides for preparing in the step 1, earlier through the distilled water wash-out, again through the 0.1mol/LNaCl eluant solution.Distilled water wash-out part gets powders A through concentrated, dialysis, alcohol precipitation after-filtration; NaCl eluant solution part gets powder B through concentrated, dialysis, alcohol precipitation after-filtration.Powders A and powder B obtain the pure component of Companumoea root polyose respectively behind SepharoseCL-6B and Sephadex G-25 gel filtration chromatography, called after CJP-1 and CJP-2 (seeing Fig. 2 for details).
Two, the purity of polysaccharide component, physico-chemical property, spectroscopic analysis, gas chromatographic analysis and molecular weight
Polysaccharide component C JP-1 and CJP-2 be through conventional method such as permethylated, all-hydrolytic, and molecular weight determination, nucleus magnetic resonance, chemistry such as infrared spectra and spectrographic technique prove polysaccharide structures.
(1) conclusive evidence of polysaccharide component purity:
Is moving phase with the Companumoea root polyose component C JP-1 and the CJP-2 of above-mentioned acquisition with 0.2mol/LNaCl solution, flow velocity is that 0.52ml/min carries out purity check through Sepharose CL-6B gel filtration chromatography, recording polysaccharide component C JP-1 is single to becoming the peak, so CJP-1 and CJP-2 are single pure product with CJP-2.
(2) physico-chemical property of Companumoea root polyose component
CJP-1 is white crystals, and is hard, easily the moisture absorption.Be not soluble in cold water, be soluble in hot water, be insoluble to organic solvents such as high concentration ethanol, acetone, propyl carbinol, dissolve rear solution fully and be transparence.The reaction of iodine-potassiumiodide is negative, and illustrate not starch-containingly, and the sulfuric acid phenol reactant is positive, and expression has the sugar existence.
CJP-2 is the white powder solid, and easily the moisture absorption is not soluble in cold water, very easily is dissolved in hot water, is insoluble to organic solvents such as high concentration ethanol, acetone, propyl carbinol; Dissolve the rear solution shape that is translucent fully, iodine-potassiumiodide reaction is negative, and illustrate that no starch exists, and the sulfuric acid phenol reactant is positive, and expression has sugared existence.
(3) UV spectrum and Infrared spectroscopy
Ultraviolet spectral analysis, the aqueous solution scans in 200~400nm interval; Infrared spectroscopy, sample thief 1mg, the KBr compressing tablet is at 450~4000cm
-1Infrared scan is carried out in the interval.
The result: infrared spectra shows that two kinds of Companumoea root polyose CJP-1, CJP-2 are the stretching vibration of OH at the broad peak that 3386cm-1,3395cm-1 occur respectively, and absorption peak occurring at 1030cm-1,1032cm-1 place is the C-O stretching vibration; Absorption peak in 2929cm-1 and the appearance of 2927cm-1 place is the C-H absorption peak, at 874cm-1 and 818cm-1 place characteristic absorbance is arranged all, illustrates to have mannoside; UV spectrum shows that the two kinds of polysaccharide in 190~400nm ultraviolet region there is no tangible absorption peak.
(4) gas chromatographic analysis
Polysaccharide after the hydrolysis of trifluoracetic acid hydrolysis method, is used sodium borohydride reduction, and pyridine and the laggard promoting the circulation of qi of aceticanhydride acetylize be chromatographic determination mutually.
The result: the retention time by standard monose gas chromatogram as can be known: CJP-1 monose consists of semi-lactosi, seminose, glucose, and their mol ratio is 1: 3.1: 6.4; CJP-2 monose consists of wood sugar, rhamnosyl, pectinose, seminose, semi-lactosi, glucose, and their mol ratio is 1: 1.6: 3.7: 1.7: 6.4: 14.0.
(5) mensuration of polysaccharide component molecular weight
Chromatographic column is AgilentPLaquagel-OHMIXED8 μ m, 7.5 * 300mm, and moving phase is the acetate buffer of 0.2mol/L, and flow velocity is 1.0ml/min, and the polysaccharide molecular weight standard is a Pullulan series.The typical curve that retention time by standard substance obtains the logarithm mapping of known standard molecular weight.To the mensuration of branch reservation time of Companumoea root polyose, can try to achieve corresponding molecular weight from typical curve, promptly CJP-1 is 3923, CJP-2 is 7215.
Three, the preparation of Companumoea root polyose derivative
1, the sulfation of Companumoea root polyose: at ambient temperature, in the anhydrous pyridine, after single holosaccharide and the chlorsulfonic acid reaction, through the alkali neutralization, dialysis, freeze-drying can get the sulfation Companumoea root polyose.
2, the carboxymethylation of Companumoea root polyose: in the NaOH aqueous solution, behind single holosaccharide and the chloroacetate reaction, through the acid neutralization, dialysis, freeze-drying can get the sulfation Companumoea root polyose.
Four, the nerve growth factor activity of Root of Java Campanumoea Crude polysaccharides, the pure component C JP-1 of polysaccharide and CJP-2
The Companumoea root polyose that obtains by technological method of the present invention is carried out biological activity test.
(1) method:
Get and pass above PC12 cell of 5 generations, after tryptic digestion disperses, make 2 * 10
5The cell suspension of individual cell/ml density is inoculated in the 35mm plastic culture dish that scribbles the calf hide glue, and every ware 2ml places 36 ℃, contains 10%CO
2Incubator in cultivate.Nutrient solution is made up of 85%DMEM, 5% tire oxen and horses serum, 10% horse serum, glutamine 0.10g/L.And be divided into each group of control group and medicine group (drug level 20 μ g/ml, 10 μ g/ml and 5 μ g/ml) by experiment.Cultivated the 8th day.That take out to cultivate respectively organizes the PC12 cell, under inverted phase contrast microscope, observe the growth and differentiation situation (the short Differentiation a little less than ± expression has the PC12 cell of PC12 cell respectively, the obviously short Differentiation of+expression, ++ the significantly short Differentiation of expression, ++ the very obviously short Differentiation of+expression, the no effect of-expression).
(2) result: find by short Differentiation activity research to neurocyte (PC12 cell), the Root of Java Campanumoea Crude polysaccharides that obtains according to technology provided by the invention has certain short PC12 cytodifferentiation effect, when being further purified through Crude polysaccharides, the holosaccharide component C JP-l and the CJP-2 that prepare have significant promotes growth, Differentiation to the PC12 cell, the growth of spinous process axle can be promoted, when concentration 10 μ g/ml, ganglionic extension (Fig. 3) can be induced.This effect is similar with the effect of nerve growth factor, is equivalent to nerve growth factor at least.(the results are shown in Table 1).
The short Differentiation of table 1 pair PC12 cell is observed
Group drug level (μ g/ml) is urged the Differentiation remarks |
NGF organizes 20ng/ml +++experimental example 1 20 ± thick polysaccharide 10-5-experimental example 2 20+CJP-1 15+10+5 ± experimental example 3 20+CJP-2 15+10 ± 5- |
Experimental results show that by a series of: Companumoea root polyose provided by the invention has the nerve growth factor effect to neurocyte, also can be to H
2O
2The neural cell injury that causes etc. chemical substance has protection and repair.Companumoea root polyose provided by the invention can be applicable to the medicine of diseases such as preparation prevention and treatment nerve degenerative diseases such as senile dementia, learning memory disorder, children mental retardation, apoplexy and is improving memory dysfunction; promote or the raising learning and memory, in the protective foods as cerebral protective agent.Companumoea root polyose also can be used as the relevant experiment of growth, differentiation and injury repairing that positive control (reagent) is used for neurocyte.
Compared with prior art, beneficial effect of the present invention is:
1) Companumoea root polyose provided by the invention and derivative are active polysaccharide new, that do not report.
The technology of 2) extraction provided by the invention, separation, purifying Companumoea root polyose can select the Guizhou Province to produce raw material or other local raw material that produces as required, prepares needed polysaccharide product easily.
3) Companumoea root polyose process for purification provided by the invention is practical, is grasping simplely, is suitable for the expansion scale, is easy to realize the purpose of industrialization.
4) Companumoea root polyose provided by the invention has the nerve growth factor effect to neurocyte (PC12 cell), and this result of study is not appeared in the newspapers as yet.Companumoea root polyose can be applicable to the medicine of diseases such as preparation prevention and treatment nerve degenerative diseases such as senile dementia, learning memory disorder, children mental retardation, apoplexy and is improving memory dysfunction; promote or the raising learning and memory, in the protective foods as cerebral protective agent.
Description of drawings: accompanying drawing 1: Root of Java Campanumoea Crude polysaccharides preparation technology schema; Accompanying drawing 2: preparation technology's schema of pure component C JP-1 of Root of Java Campanumoea and CJP-2; Accompanying drawing 3: the Differentiation synoptic diagram of the short PC12 cell of pure component C JP-1 of Root of Java Campanumoea and CJP-2.
Embodiment:
Embodiments of the invention 1: the preparation of Root of Java Campanumoea Crude polysaccharides
To take by weighing 200g and put in the round-bottomed flask with 60 ℃ of dry 2h of the Root of Java Campanumoea granulated slag behind 95% alcohol heating reflux, add sherwood oil 500ml, 60~90 ℃ of backflow 1h filter with degreasing, and sherwood oil reclaims.Filter residue eliminates solvent, filters then, and after dregs of a decoction adding distil water 1000ml soaked 1h, 40min was got in 90 ℃ of warm lixiviates, filters, and soaks 60min with 70 ℃ of temperature of 500ml distilled water again, filters, and merging filtrate is evaporated to 150ml, extracts to remove deproteinize with the Sevag method.Add 1% decolorizing with activated carbon 2 times, suction filtration, filtrate adds 95% ethanol to be made and contains alcohol amount and reach 80%, the refrigerator standing over night.Filter, residue is successively used 95% ethanol, and dehydrated alcohol, ether, acetone is repeatedly washing successively, and 60 ℃ of oven dry promptly get and make with extra care Companumoea root polyose (50g).
Embodiments of the invention 2: the preparation of holosaccharide component C JP-1
Get Crude polysaccharides 5.0g, be dissolved in the 120ml distilled water, carry out the DEAE cellulose chromatography, flow velocity 1ml/min carries out wash-out with distilled water earlier, till no elution peak.Carry out gradient elution till do not have elution peak and occur with 1mol/LNaCl for upper limit solution then.Elutriant is surveyed absorbancy with phenol sulphate reagent colour developing back.According to absorbancy effluent volume is mapped, obtain elution curve.Occur 2 main peaks altogether, collect simple spike respectively, concentrate the back dialysis, precipitate, get the 1JP-1 and the purer product of CJP-2 of white powder behind the centrifugal drying with dehydrated alcohol.
Get the purer product CJP-1 of 500mg, carry out Sepharose CL-6B gel filtration chromatography (going up sample volume 30ml, flow velocity 0.52ml/min), carry out wash-out with 0.2mol/LNaCl solution, automatic collection instrument is collected, and gets pure product CJP-1 (the phenol sulfuric acid process detects, and it is unimodal to get a symmetry).
Embodiments of the invention 3: the preparation of holosaccharide component C JP-2
Obtain purer product CJP-2 according to the method among the embodiment 2, get the purer product CJP-2 of 200mg, carry out the SepharoseCL-6B gel filtration chromatography and (go up sample volume 15ml, flow velocity 0.52ml/min), carry out wash-out with 0.2mol/LNaCl solution, automatic collection instrument is collected, and gets pure product CJP-2 (the phenol sulfuric acid process detects, and it is unimodal to get a symmetry).
Embodiments of the invention 4: the Companumoea root polyose or derivatives thereof is equipped with conventional, suitable auxiliary material, be prepared into suitable form of administration such as tablet, capsule, powder pin, injection liquid or great transfusion preparation, in treatment of diseases, the prevention such as be applied in prevention and treatment senile dementia, apoplexy, children mental retardation, memory dysfunction and go down.
Embodiments of the invention 5: the Radix Codonopsis polysaccharide or derivatives thereof that fetches earth, as foodstuff additive, form the protective foods that improves memory dysfunction, promotion or improve learning and memory, reach the effect of protection brain function.
Claims (7)
1. the preparation method of Companumoea root polyose and derivative is characterized in that: extraction from dietotherapeutic plant Root of Java Campanumoea, separation and the refining Crude polysaccharides that obtains, and CPJ-1 and two kinds of holosaccharide materials of CPJ-2 of forming it.
2. according to the preparation method of described Companumoea root polyose of claim 1 and derivative, it is characterized in that: by the piece root extraction separation Companumoea root polyose of Root of Java Campanumoea, its step is as follows:
A. the extraction of Crude polysaccharides preparation: with the piece root of Root of Java Campanumoea with reflow of alcohol removal of impurities, petroleum ether degreasing after, carry through the distillation hydro-thermal, extract can get the Root of Java Campanumoea Crude polysaccharides behind the alcohol precipitation after removing Deproteinization, decolouring again.
B. the purifying of polysaccharide is with refining: will extract the Crude polysaccharides for preparing gets Companumoea root polyose behind DEAE cellulose chromatography and Sepharose CL-6B and Sephadex G-25 gel filtration chromatography pure substance among the step a.
3. according to the preparation method of claim 1 or 2 described Companumoea root polyoses and derivative, it is characterized in that: the Companumoea root polyose that obtains is carried out sulfonated and carboxymethylation, thereby obtain the derivative of Companumoea root polyose.
4. as claim 1? in any described preparation method Companumoea root polyose and the derivative thereof that obtain.
5. as the application of Companumoea root polyose as described in the claim 4 and derivative thereof, it is characterized in that: neurocyte is had tangible promotes growth, Differentiation and can obviously repair the injured nerve cells function.
6. according to the application of described Companumoea root polyose of claim 5 and derivative thereof, it is characterized in that: Companumoea root polyose and derivative thereof are prepared into suitable form of administration, are applied in the medicine of diseases such as preparation prevention and treatment nerve degenerative diseases such as senile dementia, learning memory disorder, children mental retardation, apoplexy.
7. according to the application of described Companumoea root polyose of claim 5 and derivative thereof, it is characterized in that: Companumoea root polyose and derivative thereof are applied in preparation improve memory dysfunction, promote or improve learning and memory, in the protective foods as cerebral protective agent.
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CN101869074A (en) * | 2010-05-06 | 2010-10-27 | 浙江省农业科学院 | Tissue culture and rapid propagation method for campanumoea |
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CN101869074A (en) * | 2010-05-06 | 2010-10-27 | 浙江省农业科学院 | Tissue culture and rapid propagation method for campanumoea |
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CN115286721A (en) * | 2022-07-28 | 2022-11-04 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application of active polysaccharide composition in preparation of product with effect of preventing or treating gastric injury |
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