CN102225969A - Polysaccharide from Eucommia ulmoides leaves with anti-complement activity, method of extracting, separating and purifying the same - Google Patents
Polysaccharide from Eucommia ulmoides leaves with anti-complement activity, method of extracting, separating and purifying the same Download PDFInfo
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Abstract
The invention discloses a polysaccharide from Eucommia ulmoides leaves with anti-complement activity, and its method of extracting, separating and purifying, wherein, the polysaccharide from Eucommia ulmoides leaves with anti-complement activity is an acidic polysaccharide. The extraction method comprises the following steps: adding eucommia fragments in petroleum ether, reflowing and magnetic stirring, and extracting filtration; air drying the residues, then using hot water for boiling-refluxing extraction, filtering and removing the residues with gauze; extracting the extract, carrying out decompression condense; mixing the polysaccharide concentrate with waterless ethanol, standing for precipitation, and using centrifugation to obtain a alcohol precipitated polysaccharide. The separating and purifying method comprises the following steps: producing a polysaccharide solution from crude polysaccharide, extracting filtration, and using macroporous resin to decolour; removing protein by using Sevag method, mixing Sevag reagent with the polysaccharide sample uniformly, carrying out centrifugation, collecting supernatant and condensing, UV-scanning until no absorption peak; respectively dialyzing the crude polysaccharide solution; weighing polysaccharide to dissolve in distilled water, and eluting on a cellulose column to obtain the monomer component of refined polysaccharide. The method realizes the exploitation of folium cortex eucommiae resource, lays a foundation for the application in pharmacy industry, and serves the health of human.
Description
Technical field
The present invention relates to the polysaccharose substance of organic high molecular compound, relate in particular to the Folium Eucommiae polysaccharide, also relate to its extraction and separation purification method.
Background technology
After the sixties in 20th century, people find that gradually polysaccharose substance has special bioactive functions, find that many polysaccharide materials and derivative thereof have pharmaceutical use, especially aspect anticoagulation, antithrombotic, accent blood fat, adjusting immunologic function and antitumor, the anti-radiation significant pharmacological action and curative effect are being arranged all.On anticomplementary activity, the polysaccharide of many higher plants all has the activating complement effect, and the complement system overactivity that causes for inflammation has certain balance restraining effect.(polysaccharide from Eucommia ulmoides leaves PsEUL) is exactly a kind of in the above-mentioned polysaccharide material to the Folium Eucommiae polysaccharide.Yet, at present relevant research also seldom, and research only limits to assay, forms and pharmacological testing does not appear in the newspapers about the monose of single polysaccharide.Through retrieval, the patent application that relates to the Folium Eucommiae polysaccharide only has 1, be ZL01128743.8 number of Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology application the method for continuously extracting active " a kind of from Folium Eucommiae ", this patented technology mainly is to extract eucommia total glycosides and eucommiae total flavones, then, extract eucommia acidic polysaccharose the Eucommia ulmoides leave dregs after extracting other active ingredient.This patented technology is not separated the Folium Eucommiae polysaccharide content with purifying.And to this valuable ingredients of traditional Chinese medicine development of resources research of Folium Eucommiae, especially chemistry and the pharmacological research to the Folium Eucommiae polysaccharide has more significant meaning, certainly will cause the attention of academia.
Summary of the invention
The objective of the invention is to seek to have anticomplementary activity, have potential immunocompetent Folium Eucommiae polysaccharide, clear and definite its chemical structure by the external activity screening.
Another purpose of the present invention provides the extracting method of Folium Eucommiae polysaccharide, and the separation purification method of Folium Eucommiae polysaccharide is provided simultaneously, and lays the first stone for being applied to pharmaceutical industry.
The Folium Eucommiae polysaccharide with anticomplementary activity that the contriver screens is an acidic polysaccharose, the highest wherein active consisting of: L-rhamnosyl, D-Fucose, D-pectinose, D-wood sugar, D-glucose sugar, D-semi-lactosi and uronic acid, their massfraction is respectively: 11.8%, 1.6%, 37.7%, 4.2%, 10.7%, 12.8%, 21.2%.
The extracting method of Folium Eucommiae polysaccharide provided by the invention is dried Folium Eucommiae to be ground into＜broken group about 5mm, adds sherwood oil, and 80 ℃ are refluxed down and magnetic agitation is handled 2h, to remove surface fat and gutta-percha; Repeat once to get filter residue, suction filtration; After air-dry, use hot water extraction, add water and do not have raw material, boiling reflux extracts 2h, extracts 3 times, and three layers of gauze squeeze the filtering slag; And extracting solution, concentrating under reduced pressure is 1.1～1.2 to proportion; The polysaccharide concentrated solution is mixed quiescent setting, centrifugal 1 times of volume alcohol precipitation polysaccharide PsEUL1 with the equivalent dehydrated alcohol; Separation of supernatant, and add the long-pending ethanol of monoploid again, mixing is static, obtains 2 times of volume alcohol precipitation polysaccharide PsEUL2; The centrifugation post precipitation adds the long-pending ethanol of monoploid again in supernatant liquor, obtain 3 times of volume alcohol precipitation polysaccharide PsEUL3.
The Crude polysaccharides composition that obtains adopt Sevag method deproteinated, and the DEAE Mierocrystalline cellulose separates again through the decolouring of S-8 macroporous resin.
Above-mentioned Folium Eucommiae polysaccharide shows that through its purity of reaction assay such as sulfuric acid phynol method, fehling reagent, IKI, iron trichlorides they do not contain reducing sugar, starch, aldehydes matter; With Xylene Brilliant Cyanine G method and its protein impurities content of UV spectrophotometer measuring.
Folium Eucommiae separation of polysaccharides purification process of the present invention is: respectively Crude polysaccharides component PsEUL1, PsEUL2, PsEUL3 are configured to 2% polysaccharide soln, suction filtration is removed insoluble precipitation, with the decolouring of S-8 macroporous resin; Adopt Sevag method deproteinated, with chloroform: Sevag reagent and 1: 1 mixing of polysaccharide sample of propyl carbinol=5: 1, with the centrifugal 10min of 4000r/min rotating speed, collect supernatant liquor and concentrated, repeat to remove albumen operation 8～10 times and do not have precipitation to the middle layer, carry out UV scanning in 190～400nm, till no absorption peak; Respectively Crude polysaccharides solution being adopted molecular weight cut-off is 14000 dialysis tubing dialysis, go to zero up to the polysaccharide specific conductivity that records with conductivity meter, and till not changing; Take by weighing 0.2g Folium Eucommiae polysaccharide PsEUL1 then, be dissolved in the 3mL distilled water, the DEAE-52 of last 2.5cm * 25cm (OH) cellulose column is used 0.0 → 1mol/L NaCl eluant solution respectively, control flow velocity 1mL/min, automatically Fraction Collector is collected, and every 10mL one pipe is with sulfuric acid-phynol method pipe tracking monitor, collecting same peak contains polysaccharide and respectively manages sample, with pipe number is X-coordinate, and the polysaccharide soln absorbancy is an ordinate zou, makes elution curve.Obtain the monomer component of smart polysaccharide.
Above-mentioned PsEUL1
1, PsEUL1
2, PsEUL1
3Three smart monomers and polysaccharide components are carried out anticomplementary activity by classical pathway or alternative route and are measured.
The method that above-mentioned classical pathway is measured anticomplementary activity is: get the pH7.0～7.5 sample liquid adding test tube of 100 μ L with the phosphate buffered saline preparation; Add again 100 μ L with the dextrose gelatin veronal buffered saline by 1: 100 the dilution normal guinea pig serum and the sheep red blood cell (SRBC) (5 * 10 of 100 μ L sensitization
8Individual/mL), mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1ml cold saline termination reaction after hatching; Centrifugal 10min under the 2000r/min rotating speed gets supernatant liquor and measures absorbance value at wavelength 405nm place; Sample is calculated as follows the active inhibiting rate of complement hemolysis:
The method that above-mentioned alternative route is measured anticomplementary activity is: get the sample liquid adding test tube of 100 μ L with pH7.0～7.5 of phosphate buffered saline preparation; Add equal-volume again and (contain the 5mMC Veronal sodium with GVB-Mg-EGTA (2 *) damping fluid, 0.14mol/LNaCl, 0.1% gelatin, the 4mmol/L magnesium chloride, 16mmol/L EGTA, pH7.4), by the normal guinea pig serum of dilution in 1: 2 and 100 μ L rabbit erythrocyte suspensions (1.5 * 108/mL), mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1mL cold saline termination reaction after hatching; Centrifugal 10min under the 2000r/min rotating speed gets supernatant liquor and measures absorbance value at wavelength 405nm place; Press aforementioned calculating formula calculation sample to the active inhibiting rate of complement hemolysis.
Adopt aforesaid method that the Folium Eucommiae polysaccharide is carried out anticomplementary activity and measure, obtain following result:
The anticomplementary activity of three groups of smart polysaccharide
Can find out that smart polysaccharide group PsEUL1 and PsEUL3 have higher anticomplementary activity, and the smart polysaccharide group of PsEUL2 anticomplementary activity a little less than.
The contriver adopts sulfuric acid-carbazole method to measure the glucuronic acid content in the polysaccharide, and with vapor-phase chromatography to PsEUL1
3The composition monosaccharide groups of monomers and polysaccharide is measured.The result shows, PsEUL1
3Form by L-rhamnosyl, D-Fucose, D-pectinose, D-wood sugar, D-glucose sugar, six kinds of monosaccharide groups of D-semi-lactosi; And contain 21.2% uronic acid.
The contriver extracts the Folium Eucommiae polysaccharide from Folium Eucommiae, separation and purification obtains monomer component, and the anticomplementary activity of polysaccharide in the Folium Eucommiae studied, find the structure and the anticomplementary activity thereof of Folium Eucommiae polysaccharide first, the immunocompetent Folium Eucommiae polysaccharide that has that provides extracts and separation purification method, realized the exploitation of Folium Eucommiae resource, making it becomes possibility as the raw material of pharmaceutical industry, is the mankind's health service.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Embodiment
The extraction of 1 Folium Eucommiae polysaccharide and assay
Dried Folium Eucommiae rubbed with hand be broken into＜broken group (because of containing a large amount of Folium glue wires) about 5mm, add the sherwood oil of certain volume, 80 ℃ are refluxed and magnetic agitation is handled 2h, to remove surface fat and gutta-percha, repeat once to get filter residue, suction filtration, air-dry after, use hot water extraction, add water and do not have raw material, boiling reflux extracts 2h, extracts three times, and three layers of gauze squeeze the filtering slag, united extraction liquid, concentrating under reduced pressure is 1.1～1.2 to proportion.The polysaccharide concentrated solution is mixed quiescent setting, centrifugal 1 times of volume alcohol precipitation polysaccharide PsEUL1 with the equivalent dehydrated alcohol; Separation of supernatant, and add the long-pending ethanol of monoploid again, mixing is static, obtains 2 times of volume alcohol precipitation polysaccharide PsEUL2, and the centrifugation post precipitation adds the long-pending ethanol of monoploid again in supernatant liquor, obtain 3 times of volume alcohol precipitation polysaccharide PsEUL3.Survey its quality respectively; Survey its purity respectively with the sulfuric acid phynol method; Survey its protein content respectively with the Xylene Brilliant Cyanine G method.
2 Folium Eucommiae separation of polysaccharides purifying
Respectively Crude polysaccharides component PsEUL1, PsEUL2, PsEUL3 are configured to 2% polysaccharide soln, suction filtration is removed insoluble precipitation, with the decolouring of S-8 macroporous resin; Adopt Sevag method deproteinated, with Sevag reagent (chloroform: propyl carbinol=5: 1) with 1: 1 mixing of polysaccharide sample, centrifugal 10min (4000r/min), collect supernatant liquor and concentrated, repeat to remove albumen operation 8～10 times and do not have precipitation to the middle layer, carry out UV scanning in 190-400nm, till no absorption peak.Respectively Crude polysaccharides solution being adopted molecular weight cut-off is 14000 dialysis tubing dialysis, go to zero up to the polysaccharide specific conductivity that records with conductivity meter, and till not changing.Take by weighing 0.2g Folium Eucommiae polysaccharide PsEUL1, be dissolved in the 3mL distilled water last DEAE-52 (OH) cellulose column (2.5cm * 25cm), use 0.0 → 1mol/L NaCl eluant solution respectively, control flow velocity 1mL/min, automatically Fraction Collector is collected, and every 10mL one pipe is with sulfuric acid-phynol method pipe tracking monitor, collecting same peak contains polysaccharide and respectively manages sample, with pipe number is X-coordinate, and the polysaccharide soln absorbancy is an ordinate zou, makes elution curve.Obtain PsEUL1
1, PsEUL1
2, PsEUL1
3Three monomer components.
3 classical pathway anticomplementary activity are measured
Get sample liquid (pH7.0～7.5) the adding test tube of 100 μ L with the phosphate buffered saline preparation; Add 100 μ L again with the dextrose gelatin veronal buffered saline) the normal guinea pig serum of 1:100 dilution and the sheep red blood cell (SRBC) of 100 μ L sensitization (5 * 108/mL), mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1mL cold saline termination reaction after hatching; In the centrifugal 10min of 2000r/min, get supernatant liquor and measure absorbance value at wavelength 405nm place; Sample is to the active inhibiting rate method of calculation of complement hemolysis:
4 alternative route anticomplementary activity are measured
Get sample liquid (pH7.0～7.5) the adding test tube of 100 μ L with the phosphate buffered saline preparation; Add equal-volume again and (contain the 5mMC Veronal sodium with GVB-Mg-EGTA (2 *) damping fluid, 0.14mol/L NaCl, 0.1% gelatin, the 4mmol/L magnesium chloride, 16mmol/L EGTA, pH7.4) by the normal guinea pig serum of dilution in 1: 2 and 100 μ L rabbit erythrocyte suspensions (1.5 * 108/ml), mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1ml cold saline termination reaction after hatching; In the centrifugal 10min of 2000r/min, get supernatant liquor and measure absorbance value at wavelength 405nm place; Calculation sample is to the active inhibiting rate of complement hemolysis as stated above.
5 Folium Eucommiae active polysaccharide physico-chemical property and chemical structures
The PsEUL1 component has been carried out solvability mensuration; The fehling reagent reaction; The IKI reaction; The Molish reaction; Ferric chloride reaction; The detection of impurity such as albumen.
To monomer polysaccharide PsEUL1
2And PsEUL1
3Carrying out uronic acid measures.Precision takes by weighing polysaccharide PsEUL1 respectively
2, PsEUL1
3Each 10mg adds dissolved in distilled water, and being mixed with concentration is 100ug/mL solution.Get each 1mL of need testing solution, measure its absorbance at the 530nm place respectively, make parallel repetition sample simultaneously and measure by the typical curve method, and the content of uronic acid in the calculation sample.According to known galacturonic acid content and at 530nm place absorbance, do to such an extent that the galacturonic acid typical curve is: Y=0.0076X-0.0005, r
2=0.992, correlation coefficient r=09959[X: galacturonic acid content (μ g/mL); Y: the absorbance of mensuration],
Polysaccharide PsEUL1
3Monose form and assay.Adopt capillary gas chromatography (GC) analysis.Gas chromatograph is GC-2014 type gas chromatograph (day island proper Tianjin), hydrogen flame (FID) detector, SFAT fused-silica capillary column.The Polysaccharides sample; The preparation internal standard substance; Preparation monose reference substance sugar nitrile acetyl derivatives; Preparation sample sugar nitrile acetyl derivatives.Temperature programming: 180 ℃ to 250 ℃, 5 ℃/min of temperature rise rate keeps 1min for 180 ℃, and 250 ℃ keep 30min.Chromatographic condition: carrier gas N2 flow velocity is 1.5ml/min, and tailing air-blowing 20ml/min, H2 flow velocity are 40ml/min, and air velocity is 450ml/min; Column temperature: 180-250 ℃.The FID temperature: 270 ℃, temperature of vaporization chamber: 250 ℃; Splitting ratio 5: 1, sample size 0.2 μ L.
Each the component anticomplementary activity screening of 6 Folium Eucommiae polysaccharide
The anticomplementary activity of three groups of smart polysaccharide
As can be known, in anticomplement classical pathway test, smart polysaccharide group PsEUL1 and PsEUL3 have higher anticomplementary activity, and the smart polysaccharide group of PsEUL2 anticomplementary activity a little less than.So the smart polysaccharide group PsEUL1 the highest to anticomplementary activity done further separation and purification.Separation through DEAE-52 (OH) cellulose column obtains three monomer components, PsEUL1
1, PsEUL1
2, PsEUL1
3These three monomer polysaccharide are further carried out anticomplementary activity to be measured.
7PsEUL1
3The monose of polysaccharide is formed and content
Adopt vapor-phase chromatography to PsEUL1
3The composition monosaccharide groups of monomers and polysaccharide is measured.At first to sample and the acetylize of internal standard substance matter, this process becomes monose with the polysaccharide acetolysls earlier, makes their formation have the volatility acylate simultaneously and is convenient to gas Chromatographic Determination.But this process for acylating has two shortcomings, has the furanose of suitable o-dihydroxy that epimerization can take place at monose C2, C3, is unfavorable for identifying accurately; Next is that acid monose can't acetylize and can't measure it.By table 6 and Fig. 8,9 as can be known, PsEUL1
3Form by L-rhamnosyl, D-Fucose, D-pectinose, D-wood sugar, D-glucose sugar, six kinds of monosaccharide groups of D-semi-lactosi.As shown in the table:
PsEUL1
3In the retention time and the relative percentage composition of each monosaccharide groups.
Claims (7)
1. the Folium Eucommiae polysaccharide that has anticomplementary activity, it is characterized in that this polysaccharide is an acidic polysaccharose, the highest wherein active consisting of: L-rhamnosyl, D-Fucose, D-pectinose, D-wood sugar, D-glucose sugar, D-semi-lactosi and uronic acid, their massfraction is respectively: 11.8%, 1.6%, 37.7%, 4.2%, 10.7%, 12.8%, 21.2%.
2. the extracting method of Folium Eucommiae polysaccharide as claimed in claim 1, it is characterized in that this method be dried Folium Eucommiae is ground into＜broken group about 5mm to be to destroy Folium glue wire, add sherwood oil, 80 ℃ are refluxed down and magnetic agitation processing 2h, to remove surface fat and gutta-percha; Repeat once to get filter residue, suction filtration; After air-dry, use hot water extraction, add water and do not have raw material, boiling reflux extracts 2h, extracts 3 times, and three layers of gauze squeeze the filtering slag; And extracting solution, concentrating under reduced pressure is 1.1～1.2 to proportion; The polysaccharide concentrated solution is mixed quiescent setting, centrifugal 1 times of volume alcohol precipitation polysaccharide PsEUL1 with the equivalent dehydrated alcohol; Separation of supernatant, and add the long-pending ethanol of monoploid again, mixing is static, obtains 2 times of volume alcohol precipitation polysaccharide PsEUL2; The centrifugation post precipitation adds the long-pending ethanol of monoploid again in supernatant liquor, obtain 3 times of volume alcohol precipitation polysaccharide PsEUL3.
3. method as claimed in claim 2 is characterized in that described Folium Eucommiae polysaccharide behind quality measurement, surveys its purity respectively with the sulfuric acid phynol method; Survey its protein content respectively with the Xylene Brilliant Cyanine G method.
4. the separation purification method of Folium Eucommiae polysaccharide as claimed in claim 1, it is characterized in that this method is: respectively Crude polysaccharides component PsEUL1, PsEUL2, PsEUL3 are configured to 2% polysaccharide soln, suction filtration is removed insoluble precipitation, with the decolouring of S-8 macroporous resin; Adopt Sevag method deproteinated, with chloroform: Sevag reagent and 1: 1 mixing of polysaccharide sample of propyl carbinol=5: 1, with the centrifugal 10min of 4000r/min rotating speed, collect supernatant liquor and concentrated, repeat to remove albumen operation 8～10 times and do not have precipitation to the middle layer, carry out UV scanning in 190～400nm, till no absorption peak; Respectively Crude polysaccharides solution being adopted molecular weight cut-off is 14000 dialysis tubing dialysis, go to zero up to the polysaccharide specific conductivity that records with conductivity meter, and till not changing; Take by weighing 0.2g Folium Eucommiae polysaccharide PsEUL1 then, be dissolved in the 3mL distilled water, the DEAE-52 of last 2.5cm * 25cm (OH) cellulose column is used clear water → 1mol/L NaCl eluant solution respectively, control flow velocity 1mL/min, automatically Fraction Collector is collected, and every 10mL one pipe is with sulfuric acid-phynol method pipe tracking monitor, collecting same peak contains polysaccharide and respectively manages sample, with pipe number is X-coordinate, and the polysaccharide soln absorbancy is an ordinate zou, makes elution curve; Obtain the monomer component of smart polysaccharide.
5. method as claimed in claim 4 is characterized in that described smart monomers and polysaccharide component carries out anticomplementary activity by classical pathway or alternative route and measure.
6. method as claimed in claim 4 is characterized in that the method for described classical pathway mensuration anticomplementary activity is: get the pH7.0～7.5 sample liquid adding test tube of 100 μ L with the phosphate buffered saline preparation; Add again 100 μ L with the dextrose gelatin veronal buffered saline by the normal guinea pig serum of dilution in 1: 100 and 100 μ L sensitization promptly 5 * 10
8The sheep red blood cell (SRBC) of individual/mL, mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1ml cold saline termination reaction after hatching; Centrifugal 10min under the 2000r/min rotating speed gets supernatant liquor and measures absorbance value at wavelength 405nm place; Sample to the active inhibiting rate of complement hemolysis by following formula:
7. method as claimed in claim 4 is characterized in that the method for described alternative route mensuration anticomplementary activity is: get the sample liquid adding test tube of 100 μ L with pH7.0～7.5 of phosphate buffered saline preparation; Add the 2 times damping fluids of equal-volume with GVB-Mg-EGTA again, the normal guinea pig serum and the 100 μ L that press dilution in 1: 2 are the rabbit erythrocyte suspension of 1.5 * 108/mL, mixing gently; 30min is hatched in 37 ℃ of water-baths, and jolting gently frequently promptly adds 1mL cold saline termination reaction after hatching; Centrifugal 10min under the 2000r/min rotating speed gets supernatant liquor and measures absorbance value at wavelength 405nm place; Sample to the active inhibiting rate of complement hemolysis by following formula:
Described damping fluid contains 5m MC Veronal sodium, 0.14mol/LNaCl, 0.1% gelatin, 4mmol/L magnesium chloride, 16mmol/L EGTA, pH7.4.
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