CN1226308C - Wolfberry fruit polysaccharide separating and purifying technology - Google Patents
Wolfberry fruit polysaccharide separating and purifying technology Download PDFInfo
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- CN1226308C CN1226308C CN 02108915 CN02108915A CN1226308C CN 1226308 C CN1226308 C CN 1226308C CN 02108915 CN02108915 CN 02108915 CN 02108915 A CN02108915 A CN 02108915A CN 1226308 C CN1226308 C CN 1226308C
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Abstract
The present invention relates to a novel wolfberry fruit polysaccharide separating and purifying technology which belongs to the field of biological engineering pharmacy. Wolfberry fruit polysaccharide (crude products) separated and extracted from fresh wolfberry fruit berries is adopted as raw material; laminar analysis is carried out on the raw material with two DEAE columns in a mode of connect in series, decolorization is carried out, and impurities are removed; a SephadexG-100 column is used for laminar analysis, sample introduction and elution, and a pump which has constant quantity and high pressure (which is not more than 5MPa) is used; during the laminar analysis of the column, double distilled water is used as eluent, and a preparation column is enlarged to have an industrial middle scale; in the laminar analysis process, an ultraviolet detector is used for monitoring operation, and an absorption peak occurs at a position whose wavelength is from 550mm to 560mm in an ultraviolet spectrum of a graph; the eluent is frozen in vacuum and dried in a sublimation mode, and white loosening flocculent fiber crystals which are wolfberry fruit polysaccharide are obtained. The content of LBP in the wolfberry fruit polysaccharide separated and purified by the novel technology is more than 92%, the wolfberry fruit polysaccharide contains single ingredients, and the spectrum of a graph of the absorption peak whose ultraviolet wavelength is 553.6 nm of the SephadexG-100 column is an LBP fingerprint spectrum of a graph.
Description
Technical field: the invention belongs to the biotech medicine product field.
Background technology: Chinese Pharmacopoeia later clear and definite medicinal matrimony vine of version in 1977 is the dry mature fruit of lycium barbarum Lycium barbarum L..Also claim wolfberry fruit.Medical circle is by pharmacology and clinical experimental study, think that the main biologically active substance in the lycium barbarum is lycium barbarum polysaccharide (Lycium Barbarum polysaccharida.LBP), it is a kind of nonspecific immunostimulant, energy enhance immunity function, strengthen resistance against diseases, thereby reach effects such as anti-ageing, anti-oxidant, anti-hepatitis, antitumor and anti-AIDS.
Prior art generally selects for use dry wolfberry to make raw material to the separation and purification of lycium barbarum polysaccharide LBP, extracts Wolfberry fruit polysaccharide crude product.Then, the small dimension chromatography column of applying high voltage liquid chromatograph or preparation type high pressure liquid chromatograph, " column chromatography method " of employing traditional classical is earlier with DEAE column chromatography decolouring impurity elimination.The moving phase elutriant selects for use the different alkali lye of different gradient volumetric molar concentrations to carry out wash-out, owing to above-mentioned chemical reaction reason has produced ' salt ', again with the filling Sephadex post desalination that water regain is little, particle is little, as Sephadex G-25 post; After this, again with the filling Sephhadex column chromatography for separation that water regain is big, particle is big, as posts such as Sephadex G-100, G-300.Owing to adopt the alkali lye wash-out of gradient volumetric molar concentration, at the uv-spectrogram 460nm place of ultraviolet monitor, an absorption peak appears, promptly be named as ' lycium barbarum polysaccharide ' component.So volumetric molar concentration elutriant wash-out of each different alkali lye or different gradients all can occur an absorption peak at 460nm wavelength place, i.e. lycium barbarum polysaccharide or be the lycium barbarum polysaccharide component.So domestic each seminar's separation and purification has gone out a lot of lycium barbarum polysaccharides or has been the lycium barbarum polysaccharide component, its molecular weight is also different, and putting in order of six monose compositions is also inequality with molar ratio.Generally believe that lycium barbarum polysaccharide is a complex polysaccharide.
Summary of the invention: Wolfberry fruit polysaccharide separating and purifying method.Mainly be " column chromatography method " of having broken through traditional classical.At first, raw material does not use the Wolfberry fruit polysaccharide crude product of wolfberry fruit extraction separation; Use the Fructus Lycii original juice of fresh matrimony vine berry preparation to use advanced new technologies such as " membrane sepn extraction ", " vacuum lyophilization " again and adopted, the Wolfberry fruit polysaccharide crude product of separation and Extraction is made raw material.
Technical scheme of the present invention is such: Wolfberry fruit polysaccharide crude product dissolves with bi-distilled water, and it is added No. 1 DEAE post, normal pressure constant chromatography, decolouring, decon, UV-detector monitoring.And then normal pressure, enter placed in-line No. 2 DEAE posts, further decolouring, decon, UV-detector monitoring; Thereafter, with chromatographic solution under≤5Mpa condition of high voltage, sampling pump control constant adds Sephadex G-100 column chromatography, again with bi-distilled water≤5MPa high pressure, sampling pump control constant wash-out, UV-detector monitoring, absorption peak occurs at uv-spectrogram wavelength 553.6nm place, collect elutriant; Vacuum freeze-drying obtains the wadding fibrous crystallization of white loose---the pure product of lycium barbarum polysaccharide LBP.
DEAE column chromatography, Sephadex G-100 column chromatography, the moving phase elutriant has been selected bi-distilled water.Very important this point be! Be that the LBP that separation and purification goes out is the determinative of single component.
1, Wolfberry fruit polysaccharide separating and purifying method is at first carried out the DEAE column chromatography, and main effect is to decolour, remove impurity.What we selected to use is the plain post of diethylamino ethyl OH-fiber type, and takes two cover series connection, chromatography decolouring better, removes impurity.Simultaneously, in order to reach the suitability for industrialized production level, through scientific experimentation, we are with the specification of preparation type DEAE post, expand the huge pillar of φ 300mm * h 1000mm gradually to from φ 16mm * h 200mm, carry out to the Wolfberry fruit polysaccharide crude product chromatography decolouring and remove impurity.
The filling of DEAE post adopts the wet method natural pressure evenly to load, and with bi-distilled water DEAE is modulated into uniform slurries, evenly filling under natural pressure.Note, occur the turbulent flow phenomenon when not wanting chromatography.All under the UV-detector monitoring, its ultraviolet absorption peak collection of illustrative plates is seen accompanying drawing one for No. 1 whole chromatography process of DEAE post; All under the UV-detector monitoring, its ultraviolet absorption peak collection of illustrative plates is seen accompanying drawing two for No. 2 whole chromatography processes of DEAE post.
2, Wolfberry fruit polysaccharide crude product carries out separation and purification with Sephadex G-100 post again after the DEAE column chromatography is sloughed pigment, impurity.The sephadex Sephadex of chromatography column filling, actual is a kind of molecular sieve, owing to contain a large amount of hydroxyls, so water-wet behavior is arranged, can become gel particles by swelling in hydrolyte.During by cancellated gel grain, small-molecule substance freely is spread in the hole of gel grain as the different substance solution of molecular size, macromolecular substance then by exclusion outside the gel grain.When the adding elutriant moved down, all size molecular substance constantly spread and exclusion in two-phase, and along with the partition ratio kd difference of different substances, then the material of maximum molecule flows out at first, and at last effusive is the elutriant that contains the smallest molecule quantity of material.So, Fractional Collections successively, the district that can obtain various molecular weight is disconnected.Then, polysaccharide is separated out or obtain polysaccharide with adding suitable organic solvent with vacuum freeze-drying method.
Because the molecular weight of lycium barbarum polysaccharide is between 9900d～88000d, and the scope of sephadex G-100 separating polyose is between molecular weight 1000d～100000d.So selecting Sephadex G-100 is suitable selection.
Wet method high pressure nitrogen pulse filling is adopted in the filling of Sephadex post.With bi-distilled water Sephadex is modulated into uniform slurries, then with the filling of high pressure nitrogen pulse fast.The baseline stability of separator column when this helps chromatographic separation; Post layer structure do not destroyed when the preparative column that installs was separated with column filler.
Sephadex column chromatography sample introduction and wash-out all will use the high-pressure pump that can control constant.Pressure-controlling is at≤5Mpa.
Whole chromatography process all in the UV-detector monitoring, owing to carried out repeatedly repeating scientific experimentation, can assert that the absorption peak collection of illustrative plates at ultraviolet wavelength 553.6nm place is the finger printing of lycium barbarum polysaccharide, sees accompanying drawing three.
3, Wolfberry fruit polysaccharide crude product is behind DEAE post, Sephadex column chromatography, and elutriant promptly obtains the wadding fibrous crystallization of white loose-lycium barbarum polysaccharide LBP through vacuum lyophilization.Vacuum freeze must satisfy the requirements as follows: meet the requirement of pharmacy medical and health conditions, and can disinfection; It is subzero below 60 ℃ that water vessel-cold-trap working temperature must keep reaching, and could guarantee its normal sublimation drying; Vacuum freeze must possess can the disinfection condition, and in dryness storehouse, there is hydraulic pressure to control vacuum seal equipment automatically, after vacuum lyophilization is finished, before the storehouse door opening, the good vacuum-sealing of LBP is preserved in container, meet the medical and health regulation with the LBP that guarantees preparation, and can under vacuum state, preserve nonhygroscopic, never degenerate.
4, the fresh matrimony vine berry of 1000kg adopts new technologies such as " membrane sepn-vacuum lyophilizations ", can extraction separation Wolfberry fruit polysaccharide crude product 24kg about; Adopt improved " DEAE post, Sephadex column chromatography-vacuum lyophilization " technology again, can separation and purification go out the LBP of 1200g.
5, show the LBP performance characteristic that the method separation and purification goes out:
1), sense organ: the wadding fibrous xln of white loose.Very easily the moisture absorption is faint yellow after the moisture absorption, caking.
2), solvability: very easily water-soluble; Be dissolved in sig water; Be insoluble to ethanol, acetone, chloroform.
3), color reaction:
(1), sulfuric acid-phenol reactant, colour developing is pale brown look, decomposes the back and becomes monose.
(2), sulfuric acid-anthrone reaction, be positive.
(3), Coomassie brilliant blue reaction, be positive.
(4), fehling reagent reaction, be negative.
(5), IKI reaction, be negative.
4), absorption peak: the lycium barbarum polysaccharide aqueous solution at uv-spectrogram wavelength 550nm～560nm place, has maximum absorption peak.In certain scope, absorption peak and lycium barbarum polysaccharide content have linear relationship.
5), angle-of-rotation measuring: the lycium barbarum polysaccharide aqueous solution, adding ethanol, to make its concentration be about 10%, centrifugal must the precipitation; Add ethanol then in the supernatant liquor again, making its concentration is 20%～25%, centrifugal as to precipitate for the second time.Twice sedimentary specific optical rotation is identical, illustrates that lycium barbarum polysaccharide is single component.
6), the gel filtration chromatography method is identified: when Sephadex G-100 column chromatography, the absorption peak at uv-spectrogram wavelength 553.6nm place is single zygomorphy peak shape.The component that lycium barbarum polysaccharide is described is single component, and collection of illustrative plates is seen accompanying drawing three.
7), the lycium barbarum polysaccharide of chromatographic separation purifying, LBP content analysis: total content: 92.41%.
(1), six monose total amounts: 84.34%.
(2), micronutrient levels: 4.48%.
(3), aminoacids content: 3.59%.
The Figure of description brief description:
Figure one, DEAE cellulose column chromatography process for the first time prepare type high pressure liquid chromatography UV-detector wavelength 553.6nm monitoring spectrum.
Figure two, DEAE cellulose column chromatography process for the second time prepare type high pressure liquid chromatography UV-detector wavelength 553.6nm monitoring spectrum.
Figure three, Sephadex G-100 gel filtration chromatography process prepare type high pressure liquid chromatography UV-detector wavelength 553.6nm monitoring spectrum.It also is the finger printing of the lycium barbarum polysaccharide of identification.
Claims (7)
1, Wolfberry fruit polysaccharide separating and purifying method, adopt improved ' column chromatography method ', ' vacuum freeze-drying method ' from Wolfberry fruit polysaccharide crude product, to isolate lycium barbarum polysaccharide LBP, it is characterized in that: the Wolfberry fruit polysaccharide crude product that adopts fresh berry separation and Extraction is a raw material, after described Wolfberry fruit polysaccharide crude product is dissolved in bi-distilled water, with placed in-line two diethylamino ethyl OH
-The plain post of fiber type carries out twice decolouring, removes impurity; Carry out separation and purification with cross-linked glucose gel Sephadex G-100 post again; And then, promptly obtain the wadding fibrous crystallization of white loose-pure product of lycium barbarum polysaccharide LBP through vacuum freeze-drying, wherein the elutriant of two kinds of column chromatographies must use bi-distilled water, described diethylamino ethyl OH
-The plain post filling of fiber type adopts the wet method natural pressure evenly to load, and described Sephadex G-100 post adopts wet method high pressure nitrogen pulse filling, and pressure requires to be controlled at≤5Mpa.
2, Wolfberry fruit polysaccharide separating and purifying method according to claim 1, it is characterized in that: raw material ' Wolfberry fruit polysaccharide crude product ' is to use the Fructus Lycii original juice of fresh matrimony vine berry preparation, extract and vacuum lyophilization through membrane sepn, obtain Wolfberry fruit polysaccharide crude product less than 100,000 molecular weight.
3, Wolfberry fruit polysaccharide separating and purifying method according to claim 1 is characterized in that: use placed in-line two diethylamino ethyl OH
-Impurity is removed in the plain post decolouring of fiber type, and preparation type DEAE post specification from φ 16mm * h200mm, progressively expands the huge pillar of φ 300mm * h1000mm to, Wolfberry fruit polysaccharide crude product is carried out the column chromatography decolouring remove impurity, to reach the scale of preparation of industrialization.
4, Wolfberry fruit polysaccharide separating and purifying novel method according to claim 1, it is characterized in that: after using DEAE column chromatography impurity elimination decolouring, carry out separation and purification with Sephadex cross-linked glucose gel G-100 again, with Sephadex post specification from φ 16mm * h200mm, progressively expand φ 100mm * h1000mm high-pressure glass column chromatography to, enlarge again and promptly use stainless steel layer to analyse post; Wet method high pressure nitrogen pulse filling is adopted in the filling of Sephadex post, earlier Sephadex G-100 is modulated into uniform slurries with bi-distilled water, the high pressure nitrogen pulse of usefulness≤5Mpa filling again.
5, Wolfberry fruit polysaccharide separating and purifying method according to claim 1, it is characterized in that: when DEAE post and Sephadex chromatography, use the UV-detector of preparation type high pressure liquid chromatograph to monitor, absorption peak appears in spectral line at 553.6nm wavelength place, can assert that the absorption peak spectrum at Sephadex G-100 column chromatography UV-detector 553.6nm wavelength place is the finger printing of lycium barbarum polysaccharide LBP.
6, Wolfberry fruit polysaccharide separating and purifying method according to claim 1, it is characterized in that: behind DEAE post and Sephadex column chromatography, again with the elutriant vacuum freeze-drying, be prepared into---the pure product of the wadding fibrous crystallization lycium barbarum polysaccharide of white loose LBP, requirement, the water vessel of vacuum freeze---cold-trap, controlled temperature must subzero below 60 ℃, could guarantee its normal sublimation drying; Vacuum freeze must possess can the disinfection condition, and has hydraulic pressure to control vacuum seal equipment automatically in dryness storehouse, meets the medical and health regulation with the LBP that guarantees preparation, and can under vacuum state, preserve nonhygroscopic, never degenerate.
7, Wolfberry fruit polysaccharide separating and purifying method according to claim 1, it is characterized in that: DEAE and Sephadex column chromatography, elutriant has been selected bi-distilled water, use the bi-distilled water wash-out can not produce hydrolytic action, the sugar chain of forming six monose of lycium barbarum polysaccharide can not interrupted, an absorption peak only appears at uv-spectrogram wavelength 553.6nm place, this peak be one with the symmetric peak of axis; It is 10% that isolated LBP aqueous solution adding ethanol makes its concentration, centrifugal must the precipitation, supernatant liquor adds ethanol again to make its concentration is 20%～25% then, the centrifugal throw out that gets, twice sedimentary specific optical rotation is also identical, has proved that isolated lycium barbarum polysaccharide is the lycium barbarum polysaccharide LBP of single component, analyze after testing: six monose total amounts 84.34%, aminoacids content 3.59%, micronutrient levels 4.48%, purity is more than 92%.
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| CN 02108915 CN1226308C (en) | 2002-04-03 | 2002-04-03 | Wolfberry fruit polysaccharide separating and purifying technology |
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| CN 02108915 CN1226308C (en) | 2002-04-03 | 2002-04-03 | Wolfberry fruit polysaccharide separating and purifying technology |
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Families Citing this family (7)
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| CN100444897C (en) * | 2006-03-06 | 2008-12-24 | 南京凯瑞尔纳米生物技术有限公司 | Nano complex eye drops containing liposoluble compound drug and its preparing method |
| CN103159864B (en) * | 2011-12-19 | 2016-03-02 | 中国科学院大连化学物理研究所 | Five kinds of polysaccharide that a kind of separation purification method of lycium ruthenicum polysaccharide and separation obtain |
| CN103342755B (en) * | 2013-06-03 | 2016-01-06 | 湖北大学 | Equal one-level part IV of lycium barbarum polysaccharide and its preparation method and application |
| CN104458985B (en) * | 2014-10-14 | 2016-04-06 | 中国药科大学 | The construction method of the polynary finger-print of LBP-X and standard finger-print thereof |
| CN104987433B (en) * | 2015-07-23 | 2017-04-12 | 中国科学院西北高原生物研究所 | Preparation method of RG-I type lycium barbarum pectin with anti-aging activity |
| CN109608557A (en) * | 2019-01-10 | 2019-04-12 | 华东理工大学 | Polysaccharides extracts Isolation and purification method, Lycium chinense glycopeptide and preparation method |
| CN109932446A (en) * | 2019-03-21 | 2019-06-25 | 苏州大学 | A kind of detection method of Lycium barbarum polysaccharide extract |
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