CN1194981C - Process for preparing carraoligose - Google Patents
Process for preparing carraoligose Download PDFInfo
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- CN1194981C CN1194981C CN 02135275 CN02135275A CN1194981C CN 1194981 C CN1194981 C CN 1194981C CN 02135275 CN02135275 CN 02135275 CN 02135275 A CN02135275 A CN 02135275A CN 1194981 C CN1194981 C CN 1194981C
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- carrageenin
- resistance
- oligosaccharide
- carraoligose
- molecular weight
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Abstract
The present invention relates to a method for preparing oligosaccharide of carrageenin, which is characterized in that the carrageenin is firstly purified and dried by a grading method of KCl, water is added, and hydrosol is prepared; a diluted sulfuric acid or a dilute hydrochloric acid is added during stirring, heating is carried out for refluxing hydrolyzation, and the hydrolyzation is stopped when the mixture is cooled to be at the room temperature; a pH value is regulated to be a neutral value, filtering, desalting, primary classification, concentration and drying are carried out, a gel column is continuously used for carrying out chromatographic separation and purification. The oligosaccharide of carrageenin prepared by the method of the present invention has the advantages of low molecular weight, high dissolvability, increased bioavailability and enhanced biological activity and pharmacological activity of virus resistance, cancer resistance, bacterium resistance, etc. A series of oligosaccharide of carrageenin of different molecular weight (polymerization degree) is obtained by the method, and the basic structure of the oligosaccharide of carrageenin is not changed; the present invention provides basic data for researching the structure-activity relationship of the carrageenin and provides candidate compounds and raw material medicines for the sieving, the research, the development and the production of polysaccharide medicines and health-care products of virus resistance, cancer resistance, bacteria resistance, etc.
Description
Technical field
The present invention relates to a kind of preparation method of carraoligose.
Background technology
Carrageenin (carrageenan) is a class sulfated polysaccharide, comprise κ-, λ-, μ-, ι-, ν-, ξ-, π-, β-, γ-, δ-, α-, ω-, types such as O-carrageenin have multiple biological activity, particularly as a class galactan sulfate, its significant antiviral activity, especially anti AIDS virus (HIV) activity has caused the world of medicine's extensive concern.As a kind of polyanion, carrageenan molecule amount size and sulfate content exert an influence to its biological activity such as antiviral and pharmacologically active.Yet present all kinds of carrageenins are excessive because of molecular weight, and its application is restricted.As everyone knows, the biological activity of polysaccharide or pharmacologically active are relevant with its primary structure, higher structure, molecular weight, solubleness etc.Therefore polysaccharide is degraded into the important means that oligose is the polysaccharide structure activity study, also is the important channel of finding and developing the carbohydrate medicine.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of carraoligose, it can satisfy the demand of prior art.
A kind of preparation method of carraoligose, earlier with carrageenin through the KCl grading purification, drying is characterized in that adding water and is made into the water-sol that concentration expressed in percentage by weight is 1-7 in 80-100 ℃, under agitation adding concentration is 0.1-0.8molL
-1Sulfuric acid or hydrochloric acid in 50-100 ℃ of back hydrolysis 1-6h, be cooled to room temperature and stop hydrolysis, adjust pH filters to neutral, desalination and classification just concentrate, drying continues and uses the gel filtration chromatography separation and purification.
With the carraoligose that method of the present invention is produced, molecular weight is low, and the solubleness height can increase its bioavailability, strengthens biological activity and pharmacologically actives such as antiviral, anticancer, antibiotic.Carrageenin and fibrinogen without degraded form insoluble mixture; Its degradation product and fibrinogen then form soluble complex, and toxicity obviously reduces than undegradable.Present method has obtained the serial carraoligose of different molecular weight (polymerization degree), and its basic structure does not change, be the carrageenin structure activity study data that provides the foundation, for screening, development, exploitation and the production of polysaccharide medicine such as antiviral, anticancer, antibiotic and healthcare products provides candidate compound and bulk drug.
Embodiment
The used carrageenin of present embodiment is a kappa-carrageenan, is Yantai Alga Industry Co.'s product.Kappa-carrageenan is a kind of in the multiple carrageenin type, by β-D-semi-lactosi-4-SO
4(β-D-G-4-SO
4 -) and α-D-3, (α-D-3 6-AG) forms the line style chain molecule by 1 → 3 and 1 → 4 glycosidic link to 6-inner ether semi-lactosi.During experiment, through KCl staging purifying, drying adds water and be made into the water-sol that concentration expressed in percentage by weight is 1-7 under 80-100 ℃ with kappa-carrageenan in elder generation.Under agitation add dilute sulphuric acid or dilute hydrochloric acid (the equal 0.1-0.8molL of both concentration
-1), 50-100 ℃ of back hydrolysis 1-6h.Be cooled to room temperature with cooling bath and stop hydrolysis, adjust pH filters to neutral.Filtrate is used the ultrafiltration process desalination, and (the ultrafiltration condition: ultra-filtration equipment is Millipore ProFlux M12, and molecular weight ranges is respectively Mw＜1,000 with ultrafiltration process classification just then; 1,000＜Mw＜3,000; 3,000＜Mw＜5,000; 5,000＜Mw＜10,000; Mw＞10,000), concentrate lyophilize.Continue and use the gel filtration chromatography separation and purification: chromatography column SephadexG-100, φ 3.4 * 100cm, Mw＞10,000; Chromatography column Sephadex G-50, φ 2.6 * 100cm, 5,000＜Mw＜10,000; Chromatography column Sephadex G-25, φ 1.6 * 80cm, Mw＜5,000; Sephadex G-15, φ 1.6 * 80cm, Mw＜3,000 (elutriant is distilled water); Chromatography column Bio-gel P-6, φ 1.6 * 80cm, (elutriant is 0.25M NH in 1,000＜Mw＜6,000
4HCO
3), mark in 0.2% blue dextran-2000 is done detects with improved phenolsulfuric acid colorimetry and full-automatic chromatographic system UV 214nm, collects the sample fraction, concentrates freeze-drying.
The sulfate radical content of the oligose that makes with the gelatin turbidimetry for Determination; Measure molecular weight (chromatographic column TSK-GEL G2000PW, the 300 * 7.5i.d.mm of each oligose with efficient gel permeation chromatography (HPGPC); Moving phase is redistilled water; Detect with differential refraction detector; Make standard substance with dextran, typical curve equation: lgMw=-0.4639t
R+ 7.2721, R
2=0.9971); Mean polymerisation degree calculation formula: Mw=383DP+23DP+17, in the formula, Mw: molecular-weight average; DP: mean polymerisation degree; 383: carrageenin disaccharide unit molecular weight; 23: the nucleidic mass of Na in the carrageenin sodium salt; 17 are equivalent to the nucleidic mass of end group-OH; Infrared spectra IR:KBr compressing tablet, Nicolet 510P FTIR Fourier transformation infrared spectrometer is measured; NMR (Nuclear Magnetic Resonance) spectrum
1H-NMR and
13C-NMR:Bruker DPX-400 nuclear magnetic resonance spectrometer is measured, solvent D
2O.
When 100 ℃ of following hydrolysis 3h, the molecular-weight average that increases its main fraction with acid concentration reduces kappa-carrageenan in dilution heat of sulfuric acid; At 0.1molL
-1100 ℃ of following hydrolysis of sulfuric acid prolong in time, and main fraction molecular-weight average descends, and during to 4h, molecular-weight average descends slowly.Use 0.1-0.5molL
-1Dilute sulphuric acid hydrolysis 1-6h, separation and purification obtains 6 carraoligose component S D-1, SD-2, SD-3, SD-4, SD-5 and SD-6 (table 1).Similarly, obtain 2 carraoligose component HD-1 and HD-2 (table 1) with the hydrochloric acid hydrolysis method.
8 carraoligose components that acid-hydrolysis method obtains are white powder, and are soluble in water, aqueous solution pH 6.30-6.85, and clarification, colourless to little Huang, close (table 1) of specific optical rotation and former carrageenin; HPGPC peak shape symmetry is single, and its molecular weight is narrow distribution.The molecular-weight average of 8 components is 1,750-11, and between 500, mean polymerisation degree DP 4-28 (table 1).The carraoligose sulfur acid base unit weight that obtains with the sulphuric acid hydrolysis method is suitable with former carrageenin, then is lower than (table 1) of former carrageenin with the sulfur acid base unit weight of hydrochloric acid hydrolysis.Experiment also shows, HD-1, and SD-1 and SD-2 only form the gel of faint intensity, and other components then can not form gel.Therefore, the intermolecular association of the carraoligose for preparing is unstable or very weak, cannot or hardly form reticulated structure.
Table 1 kappa-carrageenan acid degradation product carraoligose preparation condition and physico-chemical property
Fraction hydrolysising condition Mw
aDP
b[α]
D 20SO
4%
Kappa-carrageenan+62.8 21.6
HD-1 0.1mol·L
-1，HCl，100℃，3h 11,500 28 +60.3 16.28
SD-1 0.1mol·L
-1，H
2SO
4，100℃，3h 10,500 26 +60.6 21.02
SD-2 0.2mol·L
-1，H
2SO
4，100℃，3h 8,200 20 +59.6 21.08
SD-3 0.1mol·L
-1，H
2SO
4，100℃，4h 7,500 18 +60.6 22.10
HD-2 0.2mol·L
-1，HCl，100℃，3h 4,100 10 +65.0 12.53
SD-4 0.2mol·L
-1，H
2SO
4，100℃，3h 3,400 8 +58.2 23.01
SD-5 0.2mol·L
-1，H
2SO
4，100℃，4h 2,500 6 +58.8 23.73
SD-6 0.5mol·L
-1，H
2SO
4，100℃，5h 1,730 4 +59.7 22.00
A:Mw, molecular-weight average; B:DP, mean polymerisation degree.
Table 2 kappa-carrageenan acid degradation product carraoligose ir data (cm
-1)
Fraction 3700-3100 3000-2800 1370-1210 1150-1000 940-920 850-840 850/2920
a
Kappa-carrageenan 3416.4 2919.0 1261.3 1070.0 929.7 850.1 2.27
HD-1 3418.2 2926.1 1261.2 1068.8 928.0 845.9 1.12
2951.3
SD-1 3422.5 1257.4 1046.3 920.8 848.8 2.32
2923.0
SD-2 3415.2 2950.2 1262.4 1058.9 920.8 849.7 2.02
2956.7
SD-3 3421.8 1257.0 1045.9 921.9 848.9 2.46
2911.6
HD-2 3410.9 2928.3 1260.7 1053.3 927.1 848.4 1.02
2933.1
SD-4 3421.3 1255.7 1046.6 922.2 849.0 2.46
2896.3
SD-5 3420.5 2948.3 1256.3 1138.1 930.1 850.8 2.58
SD-6 3400.1 2908.1 1258.0 1049.7 919.9 848.2 2.41
A: functional group's ratio.
Table 3 kappa-carrageenan acid degradation product carraoligose HD-1, SD-1, SD-3 and SD-4 nuclear
The nuclear magnetic resonance spectroscopy data (
13C-NMR, D
20,100MHz, δ, ppm)
a
(1→3)-β-D-Gal . . (1→4)-α-D-Gal .
C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6
κ-OK a karaoke club 105.0 72.2 81.3 76.4 77.3 63.8 97.5 72.2 81.7 80.8 79.2 71.9
Glue
HD-1 103.7 69.9 78.8 74.2 75.4 61.7 95.1 69.9 79.6 78.6 77.1 70.0
SD-1 103.9 70.2 79.0 73.6 75.4 61.8 96.0 70.2 79.7 77.2 76.2 70.3
SD-3 105.5 71.6 79.0 74.3 75.5 63.6 96.9 71.6 80.4 78.2 77.1 71.6
SD-4 105.6 71.1 79.3 74.6 75.4 63.6 98.1 71.1 80.2 79.0 76.9 71.1
In the IR collection of illustrative plates of the carraoligose SD-1 to SD-6 that obtains with sulphuric acid hydrolysis method of the present invention, 1370-1210cm
-1The characteristic absorbance (S=O stretching vibration) of the total sulfate of place's representative, 940-920cm
-1The place represents α-D-3,6-AG characteristic absorbance, 850-840cm
-1The place represents the C of β-D-G
4-SO
4Basic identical (table 2) of characteristic absorbance (axially C-O-S stretching vibration) and former carrageenin, its peak shape, peak position and intensity are also suitable, particularly characterize the C of β-D-G
4-SO
4The absorption intensity ratio (850/2920) of relative content also with close (table 2) of former carrageenin, this and chemical analysis results similar (table 1).As seen, sulfuric acid only has Degradation to carrageenin, and its structure is not seen destruction.SD-1's to SD-6
1Substantially identical (data are unlisted) of H-NMR and former carrageenin, SD-1, SD-3, SD-4's
13C-NMR demonstrates 12 carbon resonance signals that kappa-carrageenan repeats disaccharide unit, and with chemistry value of moving close (table 3) of former each carbon atom of carrageenin, when showing sulphuric acid hydrolysis, along with acid concentration increases, hydrolysis degree is strengthened, and the molecular weight of product of acquisition reduces.This separates with the molecular weight determination result consistent with gel chromatography.
In the present invention, the peak shape and the peak position of the carraoligose HD-1 that the hydrochloric acid hydrolysis method obtains and the IR collection of illustrative plates of HD-2 and former carrageenin are similar, show when with hydrochloric acid carrageenin being degraded, and its backbone structure is not produced destruction.But HD-1 and HD-2 are at 850cm
-1A little less than the place absorbed, absorption intensity ratio 850/2920 was respectively 1.12 and 1.02, and the absorption intensity ratio 2.27 low significantly (table 2) than former carrageenin shows its C
4-SO
4Partly sloughed this and chemical analysis results match (table 1).Therefore, during hydrochloric acid hydrolysis, remove carrageenin is produced Degradation, also have the effect of desulfurization acidic group.HD-1's
13In the C-NMR spectrum, 12 carbon resonance signals are close with former carrageenin also, shows that its skeleton structure is uninfluenced.
In the polysaccharide degradation process, if hydrolysising condition is too strong, then the degradation product structure may be destroyed.Present method has obtained the carraoligose of different molecular weight (polymerization degree), and its basic structure do not change, and its primary structure still is β-D-G-4-SO
4And α-D-3,6-AG forms the line style chain molecule by 1 → 3 and 1 → 4 glycosidic link.
The used carrageenin of present embodiment is a kappa-carrageenan, and used preparation method and condition also are suitable for all carrageenins, comprising λ-, μ-, ι-, ν-, ξ-, π-, β-, γ-, δ-, α-, ω-, θ-carrageenin.
Claims (3)
1, a kind of preparation method of carraoligose, earlier with carrageenin through the KCl grading purification, drying adds water and is made into the water-sol that concentration expressed in percentage by weight is 1-7 in 80-100 ℃, under agitation adding concentration is 0.1-0.8molL
-1Sulfuric acid or hydrochloric acid, in 50-100 ℃ of back hydrolysis 1-6h, be cooled to room temperature and stop hydrolysis, adjust pH filters to neutral, desalination and classification just concentrate, drying continues and uses the gel filtration chromatography separation and purification.
2, preparation method as claimed in claim 1, it is characterized in that described carrageenin be κ-, λ-, μ-, ι-, ν-, ξ-, π-, β-, γ-, δ-, α-, ω-, θ-carrageenin.
3, preparation method as claimed in claim 1 is characterized in that described desalination and the employing of classification just ultrafiltration process.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100413509C (en) * | 2006-07-21 | 2008-08-27 | 宁波大学 | Use of lambda-carrageenan oligose in preparing blood vessel proliferation inhibitor |
| DE602007005697D1 (en) * | 2006-12-05 | 2010-05-12 | Marinomed Biotechnologie Gmbh | USE OF CARRAGEENAN FOR THE TREATMENT OF RHINOVIRUS INFECTIONS |
| CN103627752A (en) * | 2013-10-29 | 2014-03-12 | 中国海洋大学 | Method for preparing carrageenin oligosaccharides by compositely degrading eucheuma with carrageenanase and cellulase |
| CN109432005A (en) * | 2018-11-20 | 2019-03-08 | 青岛博智汇力生物科技有限公司 | A kind of allergic dermatitis spray containing marine oligosaccharide |
| CN110128561B (en) * | 2019-05-09 | 2021-07-20 | 华南理工大学 | Preparation method of asparagus functional oligosaccharide |
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