CN1273492C - Polysaccharide, preparation and use thereof - Google Patents
Polysaccharide, preparation and use thereof Download PDFInfo
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- CN1273492C CN1273492C CNB2003101065202A CN200310106520A CN1273492C CN 1273492 C CN1273492 C CN 1273492C CN B2003101065202 A CNB2003101065202 A CN B2003101065202A CN 200310106520 A CN200310106520 A CN 200310106520A CN 1273492 C CN1273492 C CN 1273492C
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The present invention relates to a polysaccharide which is composed of dextrose and uronic acid. The polysaccharide does not contain protein or nucleic acid; the molecular weight is from 1.6*10<6>Da to 2.6*10<6>Da; terminal group carbon is in alpha-conformation; the optical rotation alphaD<t> is from +131.7 degrees to +159.4 degrees. The basic framework of the polysaccharide is composed of dextrose connected by 1 to 4 glucosidic bonds; every 17 dextrose residues share one side chain which is a dextrose molecule or an uronic acid molecule; the proportion of the dextrose to the uronic acid is 2:1, and the dextrose and the uronic acid are both connected with main chain dextrose through 1 to 6 glucosidic bonds; the mass ratio of the dextrose to the uronic acid in each mol of polysaccharide is 53:1. The polysaccharide YCP of the present invention has an obvious inhibiting effect on mouse transplanted tumor Heps and S180. Thus, the polysaccharide YCP of the present invention can be applied to preparing medicines for treating tumors. The present invention also discloses a preparation method of the polysaccharide.
Description
One, technical field
The present invention relates to a kind of polysaccharide, specifically relate to a kind of ocean thalassiomycete hypoxylon polyose YCP and its method for making and purposes.
Two, background technology
Fungus polysaccharide be isolated from fungus sporophore, mycelium, fermented liquid, can control cell fission differentiation, regulate the old and feeble class active polysaccharide of cell growth.Fungus polysaccharide starts from the fifties as drug research, becomes immunopotentiating agent and cause people's interest after the sixties.Fungus polysaccharide is the immunostimulant that has more efficient to answer of generally acknowledging at present, and many fungus polysaccharide preparations have been widely used in clinical, have obtained challenging clinical effectiveness at aspects such as autoimmune disorder, immunologic hypofunction disease and tumor treatment.But at present to terrestrial organism (animal, plant, microorganism etc.) STUDY ON POLYSACHAROSE begin early, separation and purification has obtained multiple polysaccharide from these biologies, and their structure and function compared extensive studies, for example at present be used for fungus polysaccharides such as clinical lentinan, Pachymose, krestin and Schizophyllum commune Fr polysaccharides and all derived from Lu Sheng, and the marine organisms STUDY ON POLYSACHAROSE is confined to can breeding scale or the halobiontic polysaccharide that is easy to gather more.In addition, the different middle small molecules of structure type is more common in the research of marine natural product at present, the polysaccharide that most marine organisms are rich in does not give enough attention as yet, mainly concentrate on chitin, algal polysaccharides etc. [referring to Noda H, Amano H, Arashima K et al.Nippon Suisan Gakkaishi, 1989,55:1265; Zhang EX, Yu LJ, XiaoX.Chinese J Mar Drugs, 1994,13:1; Xue CH, Yu GL, Hirata T et al.Biosci Biotechnol Biochem, 1998,62:206; Liu Xiaomei, Zhang Hongquan. spirulina polysaccharide to the tumor-bearing mice chemotherapy after the influence expressed of hematopoietic cell proliferation, apoptosis and Bcl-2. Acta Pharmaceutica Sinica, 2002,37:616.], and the research report of the new active polysaccharide that marine microorganism is produced is few.
The structure of polysaccharide can be divided into one-level, secondary, three grades and quaternary structure.Its primary structure is meant composition, collating sequence and the mode of connection etc. of its monosaccharide residue; Secondary structure is meant the various polymers that form with hydrogen bonded between the polysaccharide skeletal chain, and this is related to the conformation of its molecular backbone chain, does not relate to the spatial arrangement of side chain; Tertiary structure be meant by between the hydroxyl in the saccharide residue in the polysaccharide, carboxyl, amino and other functional group by noncovalent interaction cause in order, rule and thick space conformation; Quaternary structure is meant the polysaccharide poly interchain aggregate that combination forms with noncovalent interaction power.The complete polysaccharide structures analysis of the structural analysis of polysaccharide comprises the primary structure of polysaccharide and the analysis of higher structure.In the analysis of polysaccharide primary structure, adopt traditional chemical process to combine at present mostly with physical method, the roughly feature of primary structure that can illustrate a certain polysaccharide substantially is [referring to StroopCJ, Xu Q, Retzlaff M et al.Carbohydrate research, 2002,337:335:Shashkov AS, Torgov VI, Nazarenko EL et al.Carbohydrate research, 2002,337:1119; KolenderAA, Matulewicz MC.Carbohydrate research, 2002,337:57.Yang BY, Ding Q, Montgomery R.Carbohydrate research, 2002,337:731].
We have obtained a kind of polysaccharide (being called for short YCP) and its physico-chemical property, chemical structure, biologic activity have been compared further investigation widely through extracting purifying from a strain thalassiomycetes (Hypoxylon sp.) mycelium in recent years.
Three, summary of the invention
The object of the present invention is to provide a kind of polysaccharide, its method for making and the purposes in the preparation medicine.
Concrete technical scheme of the present invention is as follows:
A kind of polysaccharide, it is made of glucose and uronic acid, does not contain protein and nucleic acid, and molecular weight is 1.6 * 10
6Da～2.6 * 10
6Da, end group carbon are α-configuration, specific rotation [α]
D t=+131.7 °～+ 159.4 °, the basic framework of polysaccharide is made of the glucose that 1 → 4 glycosidic link connects, per 17 glucosyl residues have a side chain, side chain is a glucose molecule or a uronic acid molecule, the ratio of glucose and uronic acid is 2: 1, all be connected with main chain glucose by 1 → 6 glycosidic link, glucose is 53: 1 with the ratio of the amount of substance of uronic acid in every mole of polysaccharide, and it has following structural formula:
*GlcpA on the side chain: the Glc mol ratio is 1: 2
A kind of method for making of above-mentioned polysaccharide, it is made up of the following step:
Polysaccharide YCP of the present invention is to mice-transplanted tumor Heps and S
180The obvious suppression effect is arranged, and therefore polysaccharide YCP of the present invention can be applied to prepare the medicine for the treatment of tumour.
Four, description of drawings
Fig. 1: the high-efficient liquid phase color spectral purity of polysaccharide YCP is identified collection of illustrative plates;
Fig. 2: the thin layer chromatogram of the monosaccharide component of polysaccharide YCP, wherein 1. mixing sugar, 2. rhamnosyls, 3. seminoses, 4. glucose, 5. semi-lactosi 6.YCP, 7. uronic acids;
Fig. 3: the methylation analysis step synoptic diagram of polysaccharide YCP;
Fig. 4: polysaccharide YCP Periodic acid consumption-time curve;
Fig. 5: the Infrared spectroscopy collection of illustrative plates of polysaccharide YCP;
Fig. 6: the hydrogen nuclear magnetic resonance spectrogram of polysaccharide YCP;
Fig. 7: the nuclear magnetic resonance of carbon spectrogram of polysaccharide YCP:
Fig. 8: polysaccharide YCP and Congo red complexing experiment maximum absorption wavelength curve.
Five, embodiment
Extraction, separation and purification, the evaluation of embodiment 1. polysaccharide YCP
Take by weighing wet thallus 1000g, add water and put in the tissue mincer and smash to pieces, supply water to 2000mL, 60 ℃ of water-baths were extracted 20 hours.Intermittently stir gauze elimination residue, the centrifugal precipitation of going of 2000r.p.m..Extracting solution is concentrated into 500mL, adds the chloroform of concentrated solution 1/4 volume and the propyl carbinol of 1/15 volume, thermal agitation, and standing demix removes sub-cloud organic solvent layer and middle layer, keeps water layer.Extract repeatedly 2 times.Gained solution was to tap water dialysis 24 hours.Dialyzate is concentrated into 500mL, progressively adds isopyknic ethanol, places 2 hours for 4 ℃.Centrifugal, with dehydrated alcohol, acetone, ether washing, vacuum-drying gets polysaccharide YCP crude product to precipitation successively.
Get polysaccharide YCP crude product 50mg, fully dissolving, on balance is good ion exchange column (DEAE-32), earlier with the 50mL distilled water wash, again with 0～2mol/L NaCl solution gradient wash-out, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition.Molecular sieve column on the separating obtained polysaccharide fraction of ion exchange column (Sephacryl S-400) chromatography, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition, and lyophilize promptly gets polysaccharide YCP of the present invention.
HPLC purity is identified: (8mmID * 300mm) performance liquid chromatographic column and differential refraction detector, moving phase is H to adopt Shodex SUGAR KS-805
2O, YCF1mg/ml sample introduction 20 μ l, flow velocity 1ml/min.
The results are shown in Figure 1.HPLC purity qualification result is a symmetrical peak, and the polysaccharide that shows gained is an one-component.
Extraction, separation and purification, the evaluation of embodiment 2. polysaccharide YCP
Take by weighing wet thallus 1000g, add water and put in the tissue mincer and smash to pieces, supply water to 3000mL, 80 ℃ of water-baths were extracted 12 hours.Intermittently stir gauze elimination residue, the centrifugal precipitation of going of 3000r.p.m..Extracting solution is concentrated into 1000mL, adds the chloroform of concentrated solution 1/5 volume and the propyl carbinol of 1/25 volume, thermal agitation, and standing demix removes sub-cloud organic solvent layer and middle layer, keeps water layer.Extract repeatedly 4 times.Gained solution was to tap water dialysis 36 hours.Dialyzate is concentrated into 1000mL, progressively adds 3 times of volume of ethanol, places 6 hours for 4 ℃.Centrifugal, with dehydrated alcohol, acetone, ether washing, vacuum-drying gets polysaccharide crude to precipitation successively.
Get crude product 50mg, fully dissolving, on balance is good ion exchange column (DEAE-32), earlier with the 200mL distilled water wash, again with 0～2mol/L NaCl solution gradient wash-out, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition.Molecular sieve column chromatography (Sephacryl S-400) on the separating obtained polysaccharide YCP of the ion exchange column component, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition, and lyophilize promptly gets polysaccharide YCP of the present invention, and purity is identical with embodiment's 1.
Extraction, separation and purification, the evaluation of embodiment 3. polysaccharide YCP
Take by weighing wet thallus 1000g, add water and put in the tissue mincer and smash to pieces, supply water to 4000mL, 90 ℃ of water-baths were extracted 8 hours.Intermittently stir gauze elimination residue, the centrifugal precipitation of going of 4000r.p.m..Extracting solution is concentrated into 2000mL, adds the chloroform of concentrated solution 1/7 volume and the propyl carbinol of 1/30 volume, thermal agitation, and standing demix removes sub-cloud organic solvent layer and middle layer, keeps water layer.Extract repeatedly 5 times.Gained solution was to tap water dialysis 48 hours.Dialyzate is concentrated into 2000mL, progressively adds 4 times of volume of ethanol, places 10 hours for 4 ℃.Centrifugal, with dehydrated alcohol, acetone, ether washing, vacuum-drying gets polysaccharide crude to precipitation successively.
Get crude product 50mg, fully dissolving, on balance is good ion exchange column (DEAE-32), earlier with the 300mL distilled water wash, again with 0～2mol/L NaCl solution gradient wash-out, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition.Molecular sieve column chromatography (Sephacryl S-400) on the separating obtained polysaccharide YCP of the ion exchange column component, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, and merges same composition, and lyophilize promptly gets polysaccharide YCP of the present invention, and purity is identical with embodiment's 1.
Embodiment 4.YCP weight-average molecular weight is measured
Adopt high performance liquid chromatograph, the GPC special software, chromatographic column is for surveying the special gel post ShodexSUGAR KS-805 (8mmID * 300mm), be limited to 500 * 10 on the separating ranges of polysaccharide
4Dalton, detector is a differential refraction detector.Moving phase is water: 35 ℃ of column temperatures: flow velocity 1mL/min.It is an amount of to get the different dextran molecule amount standard substance of molecular weight, makes the standardized solution that contains 1mg among every mL approximately with moving phase respectively, gets above-mentioned standardized solution 100 μ L respectively, injects liquid chromatograph, the record color atlas.By GPC special software drawing standard curve, get equation of linear regression: 1gM
w=8.948-0.4333tR (M
wKnown heavy average molecular weight for standard specimen; t
RRetention time for standard specimen).Get YCP polysaccharide 1mg, be dissolved in 1mL water, last sample 100 μ L, record color atlas.Getting 3 batch samples measures respectively.
Measurement result sees Table 1.The weight-average molecular weight of YCP polysaccharide is 1.6 * 10
6Da～2.6 * 10
6Da.
Table 1.YCP molecular weight determination
| Batch |
| 1 | 2 | 3 | |
| Molecular weight (* 10 6Da) | 1.6 | 1.9 | 2.6 |
Monosaccharide component is analyzed
The thin-layer chromatography (TLC) of embodiment 5. polysaccharide YCP
Take by weighing polysaccharide sample 5mg, place clean ampoule, add 2mol/l trifluoroacetic acid 2mL, tube sealing.100 ℃ of hydrolysis are after 12 hours, with the trifluoroacetic acid evaporate to dryness, wash 4～5 times with methyl alcohol after, use dissolved in distilled water, standby.Precision takes by weighing rhamnosyl, seminose, glucose, semi-lactosi, each 20mg of glucuronic acid, is dissolved in respectively in the 1mL distilled water, therefrom respectively draws 0.2mL solution and mixes as mixing sugar.Measure 0.75%CMC-Na solution 16mL in mortar, gradation adds the 6g silica gel G and final concentration is the NaH of 0.3mol/L
2PO
4, evenly be coated with after grinding evenly and be layered on the clean sheet glass (10 * 20cm).Leave standstill and dry, activate 1 hour at 105 ℃ before using.By propyl carbinol: acetone: water=4: 3: 1 is made into developping agent and places chromatography cylinder, puts into the plate that has activated, after saturated 10 minutes, and ascending development.After expansion finishes, take out, be chromogenic reagent with aniline-O-phthalic acid solution after, in 110 ℃ of heating 10min.
The results are shown in Figure 2.Carry out thin-layer chromatography after the complete acid hydrolysis of YCP, the glucose spot of result's clear display.
The mensuration of uronic acid and glucose content among the embodiment 6. polysaccharide YCP
The mensuration of glucuronic acid content: precision is measured standard sugar aldehydic acid solution (60 μ g/mL) 0,0.1,0.2,0.3,0.4,0.5mL is in test tube with ground stopper, supply distilled water to 0.5mL, in ice bath, dropwise add sodium tetraborate-concentrated sulfuric acid solution of 1.5mL0.0125mol/L after the precooling.Jolting mixes, and boils 5min in the boiling water-bath.Be cooled to room temperature with ice bath, every pipe add 25 μ L0.15% between hydroxyl biphenyl-0.5% sodium hydroxide solution.Shake up the back and measure photoabsorption at the 530nm place, make blank with " O " pipe, the drawing standard curve.For avoiding the interference of non-hexuronic acid composition and sodium tetraborate sulphuric acid soln reaction in the sample, can do sample contrast, hydroxyl biphenyl solution between promptly replacing with 25 μ L0.5% sodium hydroxide records absorbance value and deducts from the absorption of sample value.Get the solution that the YCP polysaccharide is made into 1mg/mL, draw 0.1mL, supply distilled water to 0.5mL, surplus operation is calculated respective concentration by regression equation again with the preparation of typical curve.
The mensuration of glucose content:
Accurately the standard sugar aldehydic acid solution of configuration 100 μ g/mL draws 0,0.1,0.2,0.4,0.6,0.8 respectively, and the 1.0mL standardized solution is put in the test tube with ground stopper, supplies distilled water to 1mL, adds 2mg/mL anthrone reagent 4mL, mixing, and boiling water bath boils 10min.The absorbancy at 620nm place is measured in the cooling back.Make blank with " O " pipe, with absorbancy to glucose concn map typical curve.
Accurately the standard glucose solution of configuration 100 μ g/mL draws 0,0.1,0.2,0.4,0.6,0.8 respectively, and the 1.0mL standardized solution is put in the test tube with ground stopper, supplies distilled water to 1mL, adds 2mg/mL anthrone reagent 4mL, mixing, and boiling water bath boils 10min.The absorbancy at 620nm place is measured in the cooling back.Make blank with " O " pipe, with absorbancy to glucose concn map typical curve.
Get the solution that the YCP polysaccharide is made into 1mg/mL, draw 0.1mL, supply distilled water to 1mL, surplus operation is with the preparation of typical curve.
Because there is certain interference in the content that the existence of uronic acid is measured glucose to the sulfuric acid anthrone method, hydroxyl biphenyl method (y=0.0064x-0.0006 between for this reason at first adopting, γ=0.9997) content of mensuration uronic acid, again according to uronic acid-sulfuric acid anthrone typical curve (y=0.0013x-0.0003, γ=0.9998) obtains the contribution of uronic acid absorbance, the absorbancy of total reducing sugar difference with it is due to the glucose colour developing, calculate the content of glucose in the total reducing sugar again according to glucose-sulfuric acid anthrone typical curve (y=0.0069x-0.0028, γ=0.9993).Can get by experiment that glucose is 975 μ g/mgYCP in the total reducing sugar, uronic acid is 12.7 μ g/mgYCP.
The gas phase analysis of the sugar alcohol acetyl derivatives of embodiment 7. polysaccharide YCP
Take by weighing polysaccharide 10mg and add the trifluoroacetic acid of 2mol/L, in ampoule, seal, in 100 ℃ of hydrolysis 8h, hydrolysate adds methyl alcohol and is dissolved in the 2mL water behind the evaporate to dryness repeatedly for 5～6 times behind the evaporate to dryness in furnace pot, add the 5mg sodium borohydride reduction, 3mg internal standard substance inositol is fully after the dissolving, place 30min, hydrolyzed solution evaporated under reduced pressure for 65 ℃.Add a small amount of strong acid ion exchange resin 732H then
+After the stirred for several minute, check that solution filters when being acidity, resin is washed 3 times repeatedly with less water, merging filtrate is evaporate to dryness in water-bath, the gained resistates adds 3mL dissolve with methanol and evaporate to dryness, repeatedly more than 4 times to remove boride, make reduzate finish-drying in little ampoul tube at last.In above-mentioned reduzate, add aceticanhydride and each 0.3mL of anhydrous pyridine; seal; in 100 ℃ of water-bath acetylize 20min, cooling is transferred in the furnace pot; add 0.5mL water; add water 2-3 time after concentrating repeatedly until evaporate to dryness, residue 1mL chloroform extraction, extracting solution is washed with 1mL; after the chloroform soln evaporated under reduced pressure, heavy molten with the 0.2mL trichloromethane again.Standard monose (D-rhamnosyl, D-pectinose, D-seminose, D-glucose, D-semi-lactosi) is handled by same procedure.
Gas phase condition is: Hewlett-Packard's 6890 gas chromatographs, HP-55% phenmethyl silane capillary column (30.0cm * 0.25mm * 0.25 μ m).Shunting-split stream sampling mouth not, 260 ℃ of injector temperatures.110 ℃ to 190 ℃ of temperature programmings, 3 ℃/min; 190 ℃ to 206 ℃, 2 ℃/min; 206 ℃ to 280 ℃, 20 ℃/min, keep 2min.Fid detector, carrier gas are N
2, flow velocity 25.0mL/min, 300 ℃ of detector temperatures.
The results are shown in Table 2.The reduction acetylate of YCP acid hydrolysis products carries out gas chromatographic analysis, YCP only occur one unimodal, with the contrast of the retention time of standard monose, can think that YCP is made up of glucose.Do not detect uronic acid in gas-chromatography, this is because uronic acid contains carboxyl, under general condition is difficult for being reduced, acetylize, gasifying the peak.
Table 2.GYCP gas chromatographic analysis
| Retention time (min) | |||||
| 23.981 | 24.358 | 31.807 | 32.128 | 32.339 | |
| D-rhamnosyl D-pectinose D-seminose D-glucose D-semi-lactosi YCP | + | + | + | + + | + |
Adjacent monosaccharide groups mode of connection is analyzed
Embodiment 8. methylation analysis
Take by weighing 20mg finish-drying sugar sample, be dissolved in 6mL anhydrous dimethyl sulfoxide (DMSO) inflated with nitrogen tube sealing, ultra-sonic oscillation 10 minutes, the NaOH of adding 200mg fine grinding, the inflated with nitrogen sealing, ultra-sonic oscillation 90min places 90min, dropwise adds CH
3I 2mL, ultra-sonic oscillation 30min places 30min, and remaining CH is removed in underpressure distillation
3I.
Subsequent step is seen Fig. 3.
Repeat above-mentionedly to methylate step once, through Infrared spectroscopy, at 3500cm
-1Near no absorption peak, show the hydroxyl exhaustive methylation on the polysaccharide.
Add 90% (V/V) formic acid 3mL in the above-mentioned sample that methylates, seal, hydrolysis 6h, reduction vaporization adds the trifluoroacetic acid of 2mol/L to doing, and seals, and in 100 ℃ of hydrolysis 8h, gas phase-mass spectrometry analysis is carried out in acetylize.
The results are shown in Table 3.
Table 3.YCP methylation reaction gas chromatography mass spectrometry result
| Methylated sugar | Mol ratio | The main fragment of mass spectrum (m/z) | Mode of |
| 2，3，4，6-Tetra-O-Me- | 0.66 16 1 | 43，45，71，87，101，117，129，145，161，205 43，45，99，101，113，117，233 43，101，117，205 | Glc-(1→ →4)-Glc-(1→ →4，6)-Glc-(1→ |
According to GC retention time (t
R) and the main fragment of MS (m/z); show in the reaction product and have 1; 5-diacetyl-2; 3; 4,6-tetramethyl-glucose; 1,4; 5-triacetyl-2; 3,6-trimethyl glucose and 1,4; 5; 6-is tetra-acetylated-2, the 3-dimethyl glucose, and its mol ratio is 0.66: 16: 1; show that 1 → 4 connects the basic framework that glucose has constituted polysaccharide; per 17 glucosyl residues have a 6-O position branch, can also infer on the side chain also to exist because of being difficult for according to this result to be reduced; acetylize and gasify and the uronic acid at peak, and the ratio of glucose in the side chain and uronic acid is 2: 1; the mol ratio of glucose and uronic acid is 53: 1 in every mole of polysaccharide molecule, and the ratio of glucose and uronic acid matches in this and the aforementioned YCP polysaccharide.
Embodiment 9 periodate oxidations and Smith degraded
With NaIO
4Be made into 15mmol/L solution, dilute 250 times, redilution becomes 10 different concns, surveys absorption value in the 223nm place, makes typical curve.
Accurately take by weighing polysaccharide YCP10mg, add 10mL NaIO
4Put 4 ℃ of dark places, or jolting, pitch time sampling dilute 250 times, in 223nm place survey absorption value, till numerical value no longer descends.Find NaIO by typical curve
4Consumption.
The NaOH storing solution 25ml of measuring 0.1mol/L adds the cold distilled water that newly boiled and is settled to 250ml.Precision takes by weighing the Potassium Hydrogen Phthalate of three parts of dry constant weights, and every part of 30mg adds the cold distilled water dissolving that 20ml newly boiled respectively, adds 2 of instructions phenolphthalein solutions after the cooling, is terminal point with NaOH standard solution titration to blush.
Behind the Periodic acid reaction terminating, with purpurum bromocresolis as indicator, with the NaOH measured in solution formic acid burst size of the 0.006576mol/L of above-mentioned demarcation.
The YCP polysaccharide adds proper amount of glycol and stirs 30min to reduce remaining Periodic acid behind 15mM Periodic acid complete oxidation.The dialysis tubing of packing into, to flowing water dialysis 48h, distill water dialysis 12h is evaporated to minimum volume in 70-80 ℃, uses NaBH
4In room temperature dark place reduction 12h, be adjusted to pH5.5 with 0.1mol/L acetate, to decompose remaining hydroborate, to distill water dialysis 24h postlyophilization.With the polysaccharide polyol of gained with the trifluoroacetic acid of 2mol/L in 100 ℃ of hydrolysis 8h, the hydrolyzed solution evaporated under reduced pressure is carried out thin-layer chromatography.
The results are shown in Figure 4.
YCP is through periodate oxidation, and 144h can react completely, and it is stable by NaIO that the consumption of Periodic acid reaches
4Typical curve (y=0.0975x-0.0064, γ=0.9997) to record the consumption of every mol polysaccharide be 1.03mol Periodic acid amount and have 0.05mol formic acid to generate, its result is identical substantially with the theoretical value of being calculated by the result that methylates 1.06 and 0.06, and methylation reaction is described, and the mol ratio between each residue of gained is correct as a result.
The Smith degraded product is tetrahydroxybutane and a small amount of glycerine is arranged through the thin-layer chromatography primary product, illustrates that YCP is main mode of connection with 1 → 4 glycosidic link, and a small amount of 1 → 6 glucose side chain that connects is arranged, this also with methylate and the Periodic acid reaction result matches.
The end group carbonoid is analyzed
Embodiment 10 polysaccharide YCP specific rotations are measured
After polysaccharide YCP weight loss on drying, precision takes by weighing 25mg, is settled to 25mL, with the membrane filtration of 0.8 μ m, gets subsequent filtrate and measures specific rotation in accordance with the law, by formula [α]
D t=α/lc calculates specific rotation [α: specific rotation; L: glass-tube length (dm); C: strength of solution (g/mL)].Getting 5 batch samples measures respectively.
Measurement result sees Table 4.Polysaccharide YCP has higher positivity specific rotation (+131.7 °～+ 159.4 °), and the end group carbon that shows YCP is α-configuration.
Table 4.YCP specific rotation is measured
| | 1 | 2 | 3 | 4 | 5 |
| Specific rotation (°) | +151.4 | +131.7 | +142.9 | +156.9 | +159.4 |
Embodiment 11 Infrared spectroscopy
Polysaccharide YCP takes by weighing 1mg after drying, scans in 4000～400cm is interval on Nicolet-170X type infrared spectrometer with pellet technique.
The infrared spectrogram of polysaccharide YCP as shown in Figure 5.At 3600～3200cm
-1A kind of broad peak occurring, is the stretching vibration of O-H.At 3000～2800cm
-1One group of peak be carbohydrate C-H stretching vibration, 1400～1200cm
-1Some peaks are angle vibrations of C-H.These two groups of characteristic absorbance that the peak is a saccharide compound.1000～1200cm
-1Between absorption peak be that stretching vibration by C-O is caused.930cm
-1Absorption be that asymmetric ring stretching vibration by D-grape pyranoid ring is caused.850cm
-1Absorption be α-anomerism the characteristic absorbance of C-H angle vibration, the end group carbon that shows polysaccharide YCP is α-configuration, this is consistent with the specific rotation measurement result.761cm
-1Absorption be that symmetrical ring stretching vibration by D-grape pyranoid ring is caused.
Embodiment 12 nuclear magnetic resonance spectroscopies
Get the polysaccharide YCP 15mg of purifying, be dissolved in deuterium for water (D
2O), on Varian 500,000,000 nuclear magnetic resonance analyser, analyze for 60 ℃.
The results are shown in Figure 6, Fig. 7.The measured NMR signal of polysaccharide YCP is as follows:
1H-NMR：δ5.37brs，4.98brs 3.97m，3.87m，3.70m，3.66m，3.43t(J＝8.6Hz).
13C-NMR：δ185.8
*(C)，100.1-100.3(CH)，77.9-78.2(CH)，75.3-75.5
*(CH)，73.6-74.0(CH)，73.1-73.4
*(CH)，71.8-72.1(CH)，70.2-70.5
*(CH
2)，70.0-70.2
*(CH
2)，61.4-61.8(CH
2)。(*: weak peak).Ownership sees Table 5.
Table 5.YCP's
13C-NMR and
1H-NMR signal ownership
| Residue | Chemical shift (ppm) | |||||
| C-1 H-1 | C-2 H-2 | C-3 H-3 | C-4 H-4 | C-5 H-5 | C-6 H-6 | |
| Glc-(1→ →4)Glc-(1→ →4，6)-Glc-(1→ GlcA-(1→ | 100.1-100.3 4.98 100.1-100.3 5.37 100.1-100.3 5.37 100.1-100.3 4.98 | 71.8-72.1 3.66 71.8-72.1 3.66 71.8-72.1 3.66 71.8-72.1 3.66 | 73.6-74.0 3.97 73.6-74.0 3.97 73.6-74.0 3.97 73.6-74.0 3.97 | 70.2-70.5 3.70 77.9-78.2 3.70 77.9-78.2 3.70 70.2-70.5 3.70 | 71.8-72.1 3.87 71.8-72.1 3.87 70.2-70.5 3.87 73.1-73.4 overlap | 61.4-61.8 3.87 61.4-61.8 3.87 70.0-70.2 overlap 185.8 - |
The repeating unit of inferring polysaccharide YCP according to above result is as follows:
*GlcpA on the side chain: the Glc mol ratio is 1: 2
Conformational analysis
Embodiment 13 KI-I
2Reaction
Compound concentration is the polysaccharide YCP solution 1mL of 1mg/mL, adds iodine reagent and (contains 0.02%I
20.2%KI solution) 0.6mL, measure the electronic absorption spectroscopy of 200～700nm.
The reaction that is negative of polysaccharide YCP and iodine reagent, UV is detected on the above no uv-absorbing of 500nm, illustrates that this polysaccharide does not have the α-Luo Xuanjiegou of starch, illustrates that this polysaccharide conformation is different from amylose starch.
Embodiment 14 Congo red experiments
Polysaccharide soln (concentration is 2mg/mL) and Congo red solution (concentration is 12.2 μ mol/L) equal-volume are mixed, leave standstill 15min, the variation of sequentially determining mixed solution maximum absorption wavelength λ max in the NaOH of 0～0.4mol/L solution.With pure Congo red solution is reference, manual length scanning.
The results are shown in Figure 9.Red shift does not take place with the maximum absorption wavelength of Congo red title complex in polysaccharide YCP in Congo red experiment, the metastable region do not occur yet, shows that there is not the multiply helical conformation in polysaccharide YCP molecule, and prompting polysaccharide YCP may exist with the random coil conformation in the aqueous solution.
Embodiment 15. polysaccharide YCP iv are to the restraining effect of mice-transplanted tumor Heps
Get ICR small white mouse (18～22g, male and female half and half) 60, press transplanted tumor organon inoculation Heps solid-type, inoculate back 24 hours and claim mouse heavy, and be divided into 6 groups at random, and blank group and Lentinan (5mg/kg) group, endoxan (20mg/kg) group are respectively the positive and negative control group, and polysaccharide YCP group is established high, medium and low three dosage groups (9,3,1mg/kg).Inoculate administration after 24 hours, the iv administration, per 2 days are once, and administration is 4 times altogether, and the 2nd day execution tumor-bearing mice weighed after drug withdrawal, and separates the knurl piece and weigh, and the gained data are carried out statistical procedures (t check).
The results are shown in Table 6.Compare with the physiological saline control group, polysaccharide YCP (9,3mg/kg) group, Lentinan group and Cy group all have the tumor growth effect of remarkable inhibition Heps, and wherein polysaccharide YCP group does not have obvious influence with the Lentinan group to weight of mice, and the Cy group has the effect of significant inhibition weight of mice.Experiment repeats 3 times, and the result is close.
Table 6 YCP iv is to restraining effect (X ± SD) (n=10) of mice-transplanted tumor Heps
| Batch | Group | Dosage (mg/kg) | Mouse body weight (g) | Knurl heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| 1 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 19.50±1.28 19.40±1.02 20.00±1.61 19.20±1.17 19.30±1.27 19.30±1.10 | 27.9±2.47 27.80±2.23 27.90±2.70 25.70±2.61 26.40±2.24 23.00±1.34 ** | 1.92±0.29 1.55±0.53 1.29±0.44 ** 1.13±0.34 ** 1.22±0.44 ** 0.62±0.19 ** | 0 18.98 32.59 40.98 36.65 67.94 |
| 2 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 19.30±1.10 19.40±1.20 19.20±1.60 19.40±1.11 19.50±1.02 19.30±1.10 | 27.60±1.50 27.70±1.49 28.00±1.26 27.70±1.10 27.80±1.25 23.10±1.04 ** | 1.47±0.32 1.21±0.47 0.97±0.37 ** 0.83±0.35 ** 0.91±0.30 ** 0.55±0.16 ** | 0 17.59 33.81 43.29 37.90 62.78 |
| 3 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 20.32±1.12 19.78±1.06 19.54±1.04 19.75±1.09 19.75±1.21 19.60±1.20 | 27.70±2.10 27.80±1.25 27.30±1.10 27.40±1.11 27.00±1.00 23.30±1.42 ** | 1.78±0.27 1.36±0.33 ** 1.10±0.24 ** 0.99±0.35 ** 1.08±0.46 ** 0.59±0.17 ** | 0 23.92 38.07 44.58 39.53 67.15 |
*P＜0.05,
*Compare with blank group P＜0.01
Embodiment 16 polysaccharide YCPiv are to mice-transplanted tumor S
180Restraining effect
Get 60 of ICR small white mouses (18～22g, male and female half and half), press transplanted tumor organon inoculation S
180Solid-type is inoculated back 24 hours and is claimed mouse heavy, and is divided into 6 groups at random, and blank group and Lentinan (5mg/kg) group, endoxan (20mg/kg) group are respectively the positive and negative control group, YCP organize establish high, medium and low three dosage groups (9,3,1mg/kg).Inoculate administration after 24 hours, the iv administration, per 2 days are once, and administration is 4 times altogether, and the 2nd day execution tumor-bearing mice weighed after drug withdrawal, and separates the knurl piece and weigh, and the gained data are carried out statistical procedures (t check).
The results are shown in Table 7.Compare with the physiological saline control group, (9,3mg/kg) group, Lentinan group and Cy group all have remarkable inhibition S to polysaccharide YCP
IHOThe tumor growth effect, wherein polysaccharide YCP group does not have obvious influence with the Lentinan group to weight of mice, and the Cy group has the effect of significant inhibition weight of mice.Experiment repeats 3 times, and the result is close.
Table 7.YCP iv is to mice-transplanted tumor S
180Restraining effect (X ± SD) (n=10)
| Batch | Group | Dosage (mg/kg) | Mouse body weight (g) | Knurl heavy (g) | Tumour inhibiting rate (%) | |
| Before the administration | After the administration | |||||
| 1 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 19.40±0.92 19.70±1.49 19.90±1.37 19.60±1.50 19.70±1.42 19.60±1.20 | 27.50±1.69 28.20±2.99 27.50±3.04 26.80±2.32 26.90±2.74 23.60±1.20 ** | 1.88±0.75 1.61±0.52 1.29±0.28 * 1.14±0.58 * 1.20±0.35 * 0.62±0.14 ** | 0 14.53 31.40 39.49 36.24 67.27 |
| 2 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 19.95±1.27 19.90±1.30 19.30±1.19 19.60±1.11 19.70±1.00 19.70±1.49 | 25.40±2.84 26.50±2.05 26.70±1.27 27.20±0.87 27.10±1.45 22.00±1.18 ** | 1.11±0.30 0.98±0.28 0.75±0.14 ** 0.63±0.30 ** 0.70±0.23 ** 0.41±0.10 ** | 0 12.08 32.10 43.10 37.33 63.21 |
| 3 | Blank YCP Lentinan Cy | 1 3 9 5 20 | 20.00±1.26 20.20±1.40 20.30±1.27 20.10±1.45 20.00±1.41 20.20±1.25 | 27.80±1.83 27.50±1.63 27.70±1.62 27.80±1.94 27.50±1.63 22.80±1.33 ** | 1.64±0.44 1.24±0.47 1.06±0.36 ** 0.87±0.21 ** 1.02±0.32 ** 0.51±0.17 ** | 0 24.34 35.35 46.97 37.80 68.68 |
*P＜0.05,
*Compare with blank group P＜0.01
Claims (3)
1. polysaccharide, it is characterized in that: it is made of glucose and uronic acid, does not contain protein and nucleic acid, and molecular weight is 1.6 * 10
6Da～2.6 * 10
6Da, end group carbon are α-configuration, specific rotation [α] D
t=+131.7 °～+ 159.4 °, the basic framework of polysaccharide is made of the glucose that 1 → 4 glycosidic link connects, per 17 glucosyl residues have a side chain, side chain is a glucose molecule or a uronic acid molecule, the ratio of glucose and uronic acid is 2: 1, all be connected with main chain glucose by 1 → 6 glycosidic link, glucose is 53: 1 with the ratio of the amount of substance of uronic acid in every mole of polysaccharide.
2. the method for making of the described polysaccharide of claim 1 is characterized in that it is made up of the following step:
Step 1. takes by weighing ocean thalassiomycete hypoxylon wet thallus 1000g, adds water and puts in the tissue mincer and smash to pieces, supplies water to 2000～4000mL, and 60～90 ℃ of water-baths were extracted 8～20 hours, intermittently stir, and gauze elimination residue, the centrifugal precipitation of going of 2000～4000r.p.m.,
Step 2. is concentrated into 500～2000mL with the extracting solution of step 1 gained, adds the chloroform of concentrated solution 1/4～1/7 volume and the propyl carbinol of 1/15～1/30 volume, thermal agitation, standing demix removes sub-cloud organic solvent layer and middle layer, keeps water layer, extract repeatedly 2～5 times
Step 3. is dialysed the aqueous solution of step 2 gained 24～48 hours to tap water, dialyzate is concentrated into 500～2000mL, the ethanol that progressively adds 1～4 times of quality, placed 2～10 hours for 4 ℃, centrifugal, with dehydrated alcohol, acetone, ether washing, vacuum-drying gets polysaccharide crude of the present invention to precipitation successively
Step 4. is got polysaccharide crude 50mg of the present invention, fully dissolving, on balance is good ion exchange column DEAE-32, earlier with 50～300mL distilled water wash, again with 0～2mol/L NaCl solution gradient wash-out, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, merge same composition, molecular sieve Sephacryl S-400 column chromatography on the separating obtained polysaccharide fraction of ion exchange column, fraction collection, sulfuric acid anthrone colorimetry is followed the tracks of and is detected, merge same composition, lyophilize promptly gets polysaccharide YCP of the present invention.
3. the application of polysaccharide according to claim 1 in preparation medicine for treating tumor thing.
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| CN111662394B (en) * | 2019-03-05 | 2022-11-04 | 中国医学科学院药物研究所 | Semi-synthesis preparation method and application of chondroitin sulfate polysaccharide |
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| CN112876576B (en) * | 2021-02-08 | 2022-12-20 | 河南农业大学 | Chinese yam bark polysaccharide and preparation method and application thereof |
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