CN103951760A - Centipede algae oligose and preparation method and application thereof - Google Patents
Centipede algae oligose and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses centipede algae oligose and a preparation method and application thereof. The centipede algae oligose has a chemical structure shown in the specification. The preparation method comprises the following steps: firstly, carrying out acidic hydrolysis on centipede alga, and then separating and purifying. The experiment proves that the centipede algae oligose G19 provided by the invention can inhibit growth of tumor cells in vitro and in vivo, but does not affect growth of normal hepatocyte. The research shows that the inhibiting effect of the centipede algae oligose G19 relates to tumor cell cycle arrest and tumor cell apoptosis. Therefore, the centipede algae oligose G19 provided by the invention has a potential value of being developed into an anti-tumor drug which is low in toxicity and high in efficiency.
Description
Technical field
The invention belongs to natural medicine technical field, specifically, relate to a kind of Grateloupia filicina (Wulf.) oligosaccharides and its preparation method and application.
Background technology
Grateloupia filicina (Wulf.) (Grateloupia filicina C.Ag.) belongs to rhodophyta, true Rhodophyceae, Cryptonemiales, Hai Mo section, centipede Trentepohlia algae, since ancient times just as a kind of ocean Chinese medicine, there is clearing heat and detoxicating and effect expelling parasite, and aboundresources, coastal in the whole nation all have distribution widely, and eaten by local people.Polysaccharide, as the main chemical compositions of Grateloupia filicina (Wulf.), be take Polygalactan sulfuric ester as main, and research both domestic and external fully shows that Grateloupia filicina (Wulf.) polysaccharide has good anticoagulation, the multiple biological activity such as antiviral, antitumor, illustrates that it has larger value of exploiting and utilizing.Compared with polysaccharide, oligosaccharides has molecular weight, simple in structure, easy to control the quality and good features such as the one-tenth property of medicine.But up to the present, have no the relevant report of relevant Grateloupia filicina (Wulf.) oligosaccharides and activity research thereof.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of Grateloupia filicina (Wulf.) oligosaccharides G19 with precise structure and its preparation method and application, to widen the application of Grateloupia filicina (Wulf.).
For achieving the above object, the technical solution used in the present invention is as follows:
A Grateloupia filicina (Wulf.) oligosaccharides, its molecular formula is C
48h
76o
52s
4na
4, by a non-reduced end of 4-sulfate-β-D-semi-lactosi and 3 Isosorbide-5-Nitrae-β-D-semi-lactosis and 3 4-sulfate-1,3-β-D-semi-lactosi alternate links forms, and with one 3,6-dehydration-Isosorbide-5-Nitrae-D-semi-lactosi is reduction end, and its chemical structure is as follows:
A method of preparing Grateloupia filicina (Wulf.) oligosaccharides of the present invention, is first Grateloupia filicina (Wulf.) polysaccharide to be carried out to acidic hydrolysis, then carries out separation and purification.
As a kind of preferred version, described preparation method specifically comprises the steps:
A) prepare Grateloupia filicina (Wulf.) polysaccharide;
B) Grateloupia filicina (Wulf.) polysaccharide step a) being obtained is first hydrolyzed at 60～100 ℃ with hydrochloric acid soln, and hydrolyzed solution, with in sodium hydroxide solution and rear concentrated, alcohol precipitation, centrifugal removal Grateloupia filicina (Wulf.) polysaccharide precipitation, is collected to supernatant liquor and is dried;
C) by step b) residue that obtains is soluble in water, carries out column chromatography, and water, 0.02～0.1mol/L NaCl solution and 1.5～2.5mol/L NaCl eluant solution, collect each several part elutriant successively;
D) by step c) the 0.05mol/L NaCl eluant solution that obtains part carries out column chromatography after desalination, then water, 0.01mol/L NaCl solution and 1.0～2.0mol/LNaCl solution carry out wash-out successively, collect elutriant, draw elution curve, press out peak sequencing 0.01mol/L NaCl eluant solution position is divided into A and B two portions;
E) by steps d) be dissolved in water after the A part elutriant that obtains is dry, then centrifuging and taking supernatant liquor carries out column chromatography, and with 0.15～0.2mol/L NaCl eluant solution, collection relative retention time is the component between 0.88～0.91;
F) by step e) gained relative retention time is 0.88～0.91 component is carried out column chromatography purification again, obtains described Grateloupia filicina (Wulf.) oligosaccharides, note by abridging as G19.
As further preferred version, step b) described in the concentration of hydrochloric acid soln be 0.01mol/L, the concentration of described sodium hydroxide solution is 2mol/L.
As further preferred version, step c) in column chromatography chromatographic column used be Q Sepharose Fast Flow reinforcing yin essence ion-exchange gel post; Steps d) in, column chromatography chromatographic column used is Sephadex G10 gel column; Step e) and step f) in column chromatography chromatographic column used be Superdex30 sephadex column.
Grateloupia filicina (Wulf.) oligosaccharides of the present invention (G19) can be used as one of activeconstituents or unique activeconstituents for the preparation of antitumor drug; Described tumour comprises neurospongioma, liver cancer, carcinoma of the pancreas, adenocarcinoma of lung etc.
The present invention's separation and purification from Grateloupia filicina (Wulf.) obtains Grateloupia filicina (Wulf.) oligosaccharides G19, and experimental results show that: Grateloupia filicina (Wulf.) oligosaccharides G19 provided by the invention can be in vitro and body in the growth of inhibition tumor cell, but do not affect the growth of normal liver cell, and research shows that this restraining effect of Grateloupia filicina (Wulf.) oligosaccharides G19 is relevant to tumour cell cycle retardance and apoptosis of tumor cells, therefore, Grateloupia filicina (Wulf.) oligosaccharides G19 provided by the invention has the potential value that is developed to the efficient antitumor drug of low toxicity.
Accompanying drawing explanation
Fig. 1 is Grateloupia filicina (Wulf.) oligosaccharides G19 feature liquid chromatogram;
Fig. 2 is Grateloupia filicina (Wulf.) oligosaccharides G19 sugar compositional analysis feature gas chromatography mass spectrometry total ion current figure;
Fig. 3 is Grateloupia filicina (Wulf.) oligosaccharides G19 absolute configuration analytical characteristic gas chromatography mass spectrometry total ion current figure, and A is G19 total ion current figure, and B is standard substance total ion current figure;
Fig. 4 is Grateloupia filicina (Wulf.) oligosaccharides G19 characteristic infrared spectrum figure;
Fig. 5 is Grateloupia filicina (Wulf.) oligosaccharides G19 feature
1h-NMR collection of illustrative plates;
Fig. 6 is Grateloupia filicina (Wulf.) oligosaccharides G19's
13c-NMR tests collection of illustrative plates, and wherein, A is G19
13c-NMR characteristic spectrum, B is Dept135 collection of illustrative plates;
Fig. 7 is Grateloupia filicina (Wulf.) oligosaccharides G19 feature 1H-1H COSY collection of illustrative plates;
Fig. 8 is Grateloupia filicina (Wulf.) oligosaccharides G19 feature HSQC collection of illustrative plates;
Fig. 9 is Grateloupia filicina (Wulf.) oligosaccharides G19 feature HMBC collection of illustrative plates;
Figure 10 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on human glioma U-87MG Growth of Cells;
Figure 11 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on human glioma A172 Growth of Cells;
Figure 12 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on human hepatoma HepG2 cell's growth;
Figure 13 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on human pancreas cancer Panc-1 Growth of Cells;
Figure 14 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on human lung adenocarcinoma A549 Growth of Cells;
Figure 15 is the impact of Grateloupia filicina (Wulf.) oligosaccharides G19 on Human normal hepatocyte LO2 growth;
Figure 16 is the restraining effect of Grateloupia filicina (Wulf.) oligosaccharides G19 to neuroglial cytoma U-87MG growth of xenografted in nude mice;
Figure 17 is that Grateloupia filicina (Wulf.) oligosaccharides G19 causes human glioma U-87MG cell-cycle arrest;
Figure 18 is Grateloupia filicina (Wulf.) oligosaccharides G19 induction human glioma U-87MG apoptosis.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The Grateloupia filicina (Wulf.) polysaccharide using in the present invention is made by the disclosed method of Chinese patent CN101851298A.
Embodiment 1: the preparation of Grateloupia filicina (Wulf.) oligosaccharides G19
1, the investigation of hydrolysising condition
Get Grateloupia filicina (Wulf.) polysaccharide 1g, be placed in 250mL Erlenmeyer flask, add 100mL distilled water, put in the water-bath of differing temps and fully dissolve under heated and stirred, add 1mol/L HCl solution 1～4mL, stir mixed even, continue at insulation hydrolysis 1 in water-bath, 2, 4 and 8h, take out, with 1～2mol/L NaOH solution, neutralize, centrifugal 10min under 4000g, get supernatant liquor, the ethanol precipitation polysaccharide that adds 300mL95v/v%, standing over night, the centrifugal 10min of 4000g, difference collecting precipitation and supernatant liquor, precipitate the postlyophilization that is dissolved in water, obtain polysaccharide part, supernatant liquor is evaporated under reduced pressure in 60 ℃ of water-baths, after being dissolved in water, resistates uses Sephadex G10 gel column desalination postlyophilization, obtain oligosaccharide part G19.Concrete hydrolysising condition and results of hydrolysis are in Table 1.Visible, with the rising of hydrolysis temperature and the prolongation of hydrolysis time and acidity increase, polysaccharide part reduces gradually, oligosaccharides part increases gradually, but temperature is during 60 ℃ of left and right or lower than 60 ℃, even if hydrolysis time reaches 8h, also only obtain a small amount of oligosaccharide part, illustrate that hydrolysis temperature is lower and be unfavorable for that polysaccharide hydrolysis is oligosaccharide.
Table 11g Grateloupia filicina (Wulf.) polysaccharide under different hydrolysising conditions, degrade gained polysaccharide and oligosaccharide yield
| Concentration of hydrochloric acid mol/L | Hydrolysis temperature/℃ | Hydrolysis time/h | Polysaccharide part/g | Oligosaccharides part/g |
| 0.01 | 37 | 1 | 0.964 | - |
| 0.01 | 37 | 2 | 0.952 | - |
| 0.01 | 37 | 4 | 0.943 | 0.015 |
| 0.01 | 37 | 8 | 0.930 | 0.023 |
| 0.01 | 61 | 1 | 0.933 | 0.018 |
| 0.01 | 61 | 2 | 0.901 | 0.032 |
| 0.01 | 61 | 4 | 0.873 | 0.08 |
| 0.01 | 61 | 8 | 0.835 | 0.105 |
| 0.01 | 76 | 1 | 0.892 | 0.081 |
| 0.01 | 76 | 2 | 0.773 | 0.182 |
| 0.01 | 76 | 4 | 0.706 | 0.226 |
| 0.01 | 76 | 8 | 0.585 | 0.307 |
| 0.01 | 100 | 1 | 0.412 | 0.362 |
| 0.01 | 100 | 2 | 0.264 | 0.476 |
| 0.01 | 100 | 4 | 0.122 | 0.535 |
| 0.01 | 100 | 8 | 0.063 | 0.613 |
| 0.02 | 80 | 2 | 0.693 | 0.247 |
| 0.02 | 80 | 4 | 0.501 | 0.368 |
| 0.02 | 100 | 1 | 0.313 | 0.556 |
| 0.02 | 100 | 2 | 0.138 | 0.604 |
| 0.04 | 80 | 2 | 0.485 | 0.331 |
| 0.04 | 80 | 4 | 0.353 | 0.403 |
| 0.04 | 100 | 1 | 0.238 | 0.452 |
| 0.04 | 100 | 2 | 0.105 | 0.588 |
2, reinforcing yin essence ion-exchange gel column separating purification
Get Q Sepharose Fast Flow type reinforcing yin essence ion-exchange gel 1L, with 0.5mol/L sodium hydroxide solution, soak 30min, on sintered filter funnel, suction filtration is removed alkali lye, add deionized water rinsing filtration repeatedly, until filtrate is neutral, then add 0.5mol/L salt acid soak 30min, suction filtration is removed acid solution, add deionized water rinsing filtration repeatedly, until filtrate is neutral, add appropriate amount of deionized water suspendible gel and be pulpous state, carefully pack in 5 * 60cm chromatographic column, natural subsidence is to post bed surface-stable, then connect constant flow pump, deionized water rinsing balance chromatographic column with 2 times of column volumes.
By above-mentioned 60～100 ℃ of Water Under solution gained Grateloupia filicina (Wulf.) oligosaccharides G19 crude product 5g 20mL deionized water dissolvings, centrifugal 10min under 10000g, get supernatant liquor and be splined on above-mentioned chromatographic column, water, 0.05mol/L NaCl solution and 2.0mol/L NaCl eluant solution successively, Fraction Collector is collected elutriant, phenolsulfuric acid method is followed the tracks of and is detected, and collects each several part elutriant.
Get 0.05mol/L NaCl eluant solution position solution, 60 ℃ of water-baths are evaporated to about 50mL, centrifugal 10min under 10000g, gets Sephadex G-10 sephadex column (3.5 * 60cm) desalination for supernatant liquor, obtains 0.05mol/L NaCl eluant solution position oligosaccharide solution.
Get 0.05mol/LNaCl eluant solution position oligosaccharide solution after desalination, again be splined on Q Sepharose Fast Flow ion-exchange gel post, water, 0.01mol/L NaCl solution and 2mol/L NaCl eluant solution successively, Fraction Collector is collected elutriant, phenolsulfuric acid method detects, draw elution curve, according to elution curve, press out peak sequencing 0.01mol/L NaCl eluant solution position is divided into two portions A and B.
3, Superdex30 dextrane gel column separating purification
Get above-mentioned 0.01mol/L NaCl eluant solution gained A part solution, 60 ℃ of water-bath evaporated under reduced pressure, then add 5mL water dissolution, centrifugal 10min under 10000g, get supernatant liquor and be splined on Superdex30 sephadex column (2.6 * 100cm), take 0.2mol/L NaCl solution as the separation and purification of moving phase wash-out, differential detector detects online, take salt peak in elution curve is reference, and collecting relative retention time is the chromatographic peak between 0.88～0.91.
Getting relative retention time is chromatographic peak elutriant between 0.88～0.91,60 ℃ of water-bath evaporated under reduced pressure, adding the abundant suspendible of 5mL deionized water dissolves, the centrifugal 10min of 10000g, gets supernatant liquor and is again splined on Superdex30 sephadex column, and 0.2mol/L NaCl solution is the separation and purification of moving phase wash-out, 60 ℃ of water-baths of elutriant are evaporated to 10mL, with the desalination of Sephadex G-10 sephadex column, lyophilize, obtain described Grateloupia filicina (Wulf.) oligosaccharides G19.
The Grateloupia filicina (Wulf.) oligosaccharides G19 purity obtaining according to above-mentioned preparation process is not less than 95%.Its feature spectrogram in Sugar KS-802 glycan analysis dedicated columns as shown in Figure 1, its chromatographic condition is: moving phase: 0.2mol/L NaCl, and column temperature: 40 ℃, flow velocity: 0.8mL/min, Agilent1100 liquid chromatograph, detector: differential detector (RID).
Embodiment 2: the Structural Identification of Grateloupia filicina (Wulf.) oligosaccharides G19
1, sugared compositional analysis
Get Grateloupia filicina (Wulf.) oligosaccharides G19 sample 1mg, be placed in clean tube (1.5 * 15cm), the 4-methylmorpholine borine solution and the 200 μ L3mol/L trifluoroacetic acid solution that add the new preparation of 50 μ L, 80 ℃ of oil bath heating hydrolysis 5min, add 50 μ L4-methylmorpholine borine solution, 120 ℃ of oil bath hydrolysis 1h, hydrolyzed solution proceeds in 25mL pear shape bottle, add 100 μ L4-methylmorpholine borine solution, then add 1～2mL acetonitrile, 50～60 ℃ of water-bath evaporated under reduced pressure 3 times (only adding acetonitrile), resistates adds diacetyl oxide and each 200 μ L of trifluoroacetic acid, 50 ℃ of water-bath acetylize 10min, add 5mL water, mix, room temperature is placed 30min, add 5mL chloroform extraction, chloroform layer distilled water wash three times, with carrying out GC-MS after anhydrous sodium sulfate drying, detect analysis.Chromatographic condition is as follows:
TRACE DSQ gas chromatograph-mass spectrometer (Thermo).
Chromatographic column: TR-5ms (Thermo), 60m * 0.25mm * 0.25 μ m.
Carrier gas: He.
Flow rate of carrier gas: 1mL/min.
Temperature programming: 140 ℃ of starting temperatures, 2 ℃/min is warming up to 198 ℃, insulation 4min, 4 ℃/min is warming up to 214 ℃, and 1 ℃/min is warming up to 217 ℃, insulation 4min, 3 ℃/min is warming up to 250 ℃, insulation 5min.
Injector temperature: 250 ℃.
Transmission line temperature: 250 ℃.
Sampling volume: 1 μ L.
Mass spectrum condition:
Ion source: EI; Ion source temperature: 250 ℃; Scanning of the mass spectrum scope: 40～500.
Detected result: described Grateloupia filicina (Wulf.) oligosaccharides G19 is by semi-lactosi and a small amount of 3, and 6-Anhydrogalactose forms, and the mol ratio that area normalization method records semi-lactosi and 3,6-Anhydrogalactose is 5:1.As shown in Figure 2, wherein retention time 32.10min chromatographic peak is 3,6-Anhydrogalactose to its feature gas chromatography mass spectrometry total ion current figure, and retention time 43.15min chromatographic peak is 3,6-Anhydrogalactose.
2, determination of absolute configuration
Get Grateloupia filicina (Wulf.) oligosaccharides G19 sample 1mg, be placed in 10mL ampoule, add 1mL2mol/L TFA, sealing, 120 ℃ of hydrolysis 1h, hydrolyzed solution proceeds to 25mL pear shape bottle, 60 ℃ of water-bath evaporated under reduced pressure, then add methyl alcohol 1～2mL evaporate to dryness 3 times.Add respectively 30mg/mLNaBH
3cN methanol solution, methyl alcohol/Glacial acetic acid (4:1), each 25 μ L of S-(+)-1 amino-2-propyl alcohol/methyl alcohol (1:8) solution, 65 ℃ of oil bath reacting by heating 1.5h, take out, add methyl alcohol/Glacial acetic acid (5:1) solution 1～2mL, 60 ℃ of water-bath evaporated under reduced pressure 3 times, add again acetonitrile 1～2mL evaporate to dryness 2 times, resistates adds diacetyl oxide and each 200 μ L of pyridine, and 100 ℃ of oil bath reacting by heating 45min, take out, add 5mL water, mix, room temperature is placed 30min, adds 5mL chloroform extraction, chloroform layer distilled water wash 3 times, detect analysis with carrying out GC-MS after anhydrous sodium sulfate drying.Chromatographic condition is as follows:
TRACE DSQ gas chromatograph-mass spectrometer (Thermo).
Chromatographic column: TR-5ms (Thermo), 60m * 0.25mm * 0.25 μ m.
Carrier gas: He.
Flow rate of carrier gas: 1mL/min.
Temperature programming: 140 ℃ of starting temperatures, 5 ℃/min is warming up to 240 ℃, and 2 ℃/min is warming up to 250 ℃, and 1 ℃/min is warming up to 260 ℃, insulation 5min, 1 ℃/min is warming up to 264 ℃, insulation 2min, 4 ℃/min is warming up to 280 ℃, insulation 5min.
Injector temperature: 280 ℃.
Transmission line temperature: 280 ℃.
Sampling volume: 1 μ L.
Mass spectrum condition ion source: EI, ion source temperature: 250 ℃, scanning of the mass spectrum scope: 40～600.
Detected result: the semi-lactosi in described Grateloupia filicina (Wulf.) oligosaccharides G19 is D-type semi-lactosi completely.Its gas chromatography mass spectrometry feature spectrogram as shown in Figure 3, wherein A is described Grateloupia filicina (Wulf.) oligosaccharides G19 total ion current figure, B is standard substance total ion current figure, 43.13min be D-semi-lactosi, 43.39min is L-semi-lactosi, and sample chromatogram peak retention time is compared hysteresis 0.07min with D-semi-lactosi be large by sample solution concentration and experimental error causes jointly.
3, Infrared spectroscopy
Get described Grateloupia filicina (Wulf.) oligosaccharides G19 sample 1～2mg, by KBr pressed disc method, measure infrared spectra.
Detected result: in described Grateloupia filicina (Wulf.) oligosaccharides G19 infrared spectrogram as shown in Figure 4,3426cm
-1wide and strong absorption peak and 2900cm
-1, 1050-1100cm
-1absorption peak show that this compound is carbohydrate, 1259cm
-1the explanation of strong absorption peak contain sulfate, 850cm
-1absorption peak explanation sulfate be substituted in the C4 position of semi-lactosi, 919cm
-1absorption peak explanation semi-lactosi be pyranoid ring configuration.
4, ESI-MS analyzes
Get described Grateloupia filicina (Wulf.) oligosaccharides G19 sample 1mg, add after 1mL water dissolution, with ESI-MS, detect and analyze.Ion fragment peak shown in table 2 wherein under negative ion mode, detected, wherein quasi-molecular ion peak [M-Na+]-mass-to-charge ratio is 1681.1, and hence one can see that, and described Grateloupia filicina (Wulf.) oligosaccharides G19 molecular weight should be 1704.1.
Grateloupia filicina (Wulf.) oligosaccharides G19 characteristic ion fragment described in table 2.
| Fragment ion | m/e |
| [M-Na +] - | 1681.1 |
| [M-NaSO 3 -+H +-Na +] - | 1579.0 |
| [M-G4S-Na +] - | 1398.9 |
| [M-G4SG-Na +] - | 1255.2 |
| [M-G4SG-NaSO 3 -+H +-Na +] - | 1153.3 |
5, sulfate assay (barium sulfate-gelatin turbidimetry)
5.1 instrument
754 type ultraviolet-visible pectrophotometers
Before being used, all glasswares such as test tube all need with rare nitric acid washing.
5.2 reagent
Hydrochloric acid, potassium sulfate, bariumchloride, trichoroacetic acid(TCA) is analytical pure, gelatin is chemical pure, all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
5.3 solution preparations
1mol/L HCl solution preparation: measure 21mL concentrated hydrochloric acid, with being settled to after distilled water diluting in 250mL volumetric flask, obtain.
Potassium sulfate standardized solution preparation: accurately take 105 ℃ of potassium sulfate 27～28mg of drying constant weight, put in 25mL volumetric flask, constant volume after dissolving with 1mol/L HCl, obtains.
Grateloupia filicina (Wulf.) oligosaccharides G19 sample solution preparation: accurately take sample 12～15mg, be placed in 10mL ampoule, with transfer pipet, accurately add 5mL1mol/L HCl solution, sealing, 105 ℃ of heating hydrolysis 6h, take out, let cool to room temperature, get 0.8mL and put in 2ml centrifuge tube, add 0.8mL1mol/L HCl solution dilution, centrifugal 10min under 10000rpm, carefully draws supernatant liquor, standby.
0.5% gelatin solution preparation: take 2g gelatin, be placed in 500mL triangular flask, add 400mL distilled water, 60～70 ℃ of heating in water bath dissolve, and deposit in 4 ℃ of refrigerators, standby.
1%BaCl
2gelatin solution: take BaCl
22H
2o1.173g, is placed in 100mL volumetric flask, with standing 0.5% gelatin solution of displaying, dissolves and constant volume, obtains.
3% trichoroacetic acid(TCA) solution: take 7.5g trichoroacetic acid(TCA), proceed to 250mL volumetric flask after dissolving with distilled water, constant volume and get final product.
5.4 assay
Specification Curve of Increasing
Get 8 clean tube, add respectively 0,0,40,70,100,130,160,200 μ L potassium sulfate standardized solution, the 1st pipe adds 200 μ L distilled water, all the other each pipes are added 1mol/L HCl to 200 μ L, then each pipe adds 3.8mL3% trichoroacetic acid(TCA) solution, shakes up, and the 1st pipe adds 1mL0.5% gelatin solution, as blank, all the other each pipes add 1mLBaCl
2solution, shakes up, and after 15min, in 360nm, surveys absorbancy.Using potassium sulfate content or concentration as X-coordinate, and 360nm absorbancy is as ordinate zou, drawing standard curve.
Sample sulfate assay
Each sample accurately pipettes 200 μ l (4 parts), adds 3.8mL3% trichoroacetic acid(TCA) solution, shakes up, and the 1st pipe adds 1mL0.5% gelatin solution, and as sample blank, all the other each pipes add 1mL BaCl
2solution, shakes up, and after 15min, in 360nm, surveys absorbancy.By the absorbancy recording, according to typical curve, calculate sulfate content in each sample.
Detected result: recording in described Grateloupia filicina (Wulf.) oligosaccharides G19 sulfate content by aforesaid method is 23.4% (with SO
4 2-meter).Infer thus, in this Grateloupia filicina (Wulf.) oligosaccharides G19, on average every two saccharide residue unit, be connected with a sulfate group.
6, NMR analyzes
Get G19 sample 20mg, add 0.4mL heavy water and dissolve, add 2 μ L acetone and make interior mark, in Bruker Avance III 400M nuclear magnetic resonance analyser, detect, measure respectively
1h-NMR,
13c-NMR, Dept135, HMBC, HSQC, 1H-1H COSY, and utilize nuclear magnetic spectrum to confirm G19 structure, result is respectively as shown in Fig. 5, Fig. 6 A and Fig. 6 B, Fig. 7, Fig. 8, Fig. 9.
7, described Grateloupia filicina (Wulf.) oligosaccharides G19 structure composition is resolved
In sum, this Grateloupia filicina (Wulf.) oligosaccharides G19 is only comprised of D-semi-lactosi, molecular weight is 1704.1Da, prompting forms by being no more than 10 galactose units, sulfate content is 23.4%, and exist with sodium-salt form, infer that thus this oligosaccharide should be comprised of 8 sugar units, wherein on average every two sugar units, be connected with a sulfate, in mass spectrum, the regular mass-to-charge ratio of sloughing is that 426 fragment peak occurs, illustrate that this oligosaccharide take this fragment as repeating unit, by this fragment mass-to-charge ratio, be that this repeating unit of 426 suppositions is two galactose residues and a sulfate group existing with sodium salt, 850cm in infrared spectra
-1absorption peak explanation sulfate be substituted in the C4 position of semi-lactosi.With G, represent semi-lactosi, G4S represents 4-sulfate semi-lactosi, and this oligosaccharide skeleton symbol can be expressed as:
G4SG-G4SG-G4SG-G4SG (1)
Or GG4S-GG4S-GG4S-GG4S (2)
Sugar compositional analysis shows and wherein to contain on a small quantity 3, and 6-inner ether semi-lactosi, wherein only contains one 3 by molecular weight determination results presumption, 6-inner ether semi-lactosi,
13in C-NMR, within the scope of 93-100ppm, the anomeric carbon signal of sugar-free occurs, and 92.5,91.1ppm has two fignal centers, these two fignal centers belong to 3,6-inner ether semi-lactosi anomeric carbon signal, the reduction end of 3,6-inner ether semi-lactosi in oligosaccharide is described, with AG, represents 3,6-inner ether semi-lactosi, this oligosaccharide skeleton symbol can be expressed as:
G4SG-G4SG-G4SG-G4SAG
Consistent with said structure skeleton symbol (1).
In HMBC, C1 (101.9ppm) distant relation of the H3 of G4S (4.55ppm) and G, the H1 of G (4.41ppm) and the C3 (77.6ppm) of G4S also have distant relation, illustrate that G is connected with the C3 of G4S by glycosidic link, and the H1 of G4S (4.42ppm) has distant relation with the C4 (78.4ppm) of G, illustrate that G4S is connected with the C4 of G by glycosidic link.
In summary, described Grateloupia filicina (Wulf.) oligosaccharides G19 should have following structure:
Embodiment 3: the application of Grateloupia filicina (Wulf.) oligosaccharides G19 in antitumor drug
1, Grateloupia filicina (Wulf.) oligosaccharides G19 suppresses human glioma U-87MG Growth of Cells
The good human glioma U-87MG cell 100 μ L kinds of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (the DMEM)/high glucose medium (U.S. Hyclone company) that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5v/v%CO
2incubator in cultivate after 24 hours and add respectively 100 μ L G19, make Grateloupia filicina (Wulf.) oligosaccharides G19? final concentration is respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, and each concentration is established three multiple holes, continues to be placed in 37 ℃, 5%CO
2incubator in cultivate, after 48h, add 20 μ L5mg/mL MTT (U.S. Sigma company).Be placed in 37 ℃ of incubators and continue, after reaction 4h, to take out Tissue Culture Plate, sucking-off supernatant liquor, every hole adds 150 μ L DMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing.By microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: inhibiting rate=(OD control group-OD treatment group)/OD control group * 100%.Result as shown in figure 10,0.01,0.05,0.1,0.2 and 0.4mg/mL G19 can suppress the growth of human glioma U-87MG cell, inhibiting rate is respectively 12.82%, 19.45%, 31.81%, 68.95% and 96.58%, and has certain dose-dependently relation.
2, Grateloupia filicina (Wulf.) oligosaccharides G19 suppresses human glioma A172 Growth of Cells
The good human glioma A172 cell 100 μ L kinds of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (the DMEM)/high glucose medium (U.S. Hyclone company) that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5v/v%CO
2incubator in add respectively 100 μ L G19 after cultivating 24h, make final concentration be respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, each concentration is established three multiple holes, continues to be placed in 37 ℃, 5%CO
2incubator in cultivate, after 48h, add 20 μ L5mg/mL MTT (U.S. Sigma company).Be placed in 37 ℃ of incubators and continue, after reaction 4h, to take out Tissue Culture Plate, sucking-off supernatant, every hole adds 150 μ L DMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing.By microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: inhibiting rate=(OD control group-OD treatment group)/OD control group * 100%.Result as shown in figure 11,0.01,0.05,0.1,0.2 and 0.4mg/mL G19 can suppress the growth of human glioma A172 cell, inhibiting rate is respectively 14.87%, 27.39%, 37.69%, 57.68% and 96.18%, and has certain dose-dependently relation.
3, Grateloupia filicina (Wulf.) oligosaccharides G19 suppresses human hepatoma HepG2 cell's growth
The good human hepatoma HepG2 cell's 100 μ L kinds of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (the DMEM)/high glucose medium (U.S. Hyclone company) that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5v/v%CO
2incubator in cultivate after 24 hours and add respectively 100 μ L G19, make final concentration be respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, each concentration is established three multiple holes, continues at 37 ℃, 5%CO
2incubator in cultivate, after 48 hours, add 20 μ L5mg/mL MTT (U.S. Sigma company).Be placed in 37 ℃ of incubators and continue reaction after 4 hours, take out Tissue Culture Plate, sucking-off supernatant, every hole adds 150 μ L DMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing.By microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: inhibiting rate=(OD control group-OD treatment group)/OD control group * 100%.Result as shown in figure 12,0.01,0.05,0.1,0.2 and 0.4mg/mL G19 can suppress human hepatoma HepG2 cell's growth, inhibiting rate is respectively 7.03%, 19.70%, 34.13%, 69.86% and 95.72%, and has certain dose-dependently relation.
4, Grateloupia filicina (Wulf.) oligosaccharides G19 suppresses human pancreas cancer Panc-1 Growth of Cells
The good human pancreas cancer Panc-1 cell 100 μ L kinds of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (the DMEM)/high glucose medium (U.S. Hyclone company) that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5%CO
2incubator in add respectively 100 μ L G19 after cultivating 24h, make final concentration be respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, each concentration is established three multiple holes, continues at 37 ℃, 5v/v%CO
2incubator in cultivate, after 48h, add 20 μ L5mg/mL MTT (U.S. Sigma company).Be placed in 37 ℃ of incubators and continue, after reaction 4h, to take out Tissue Culture Plate, sucking-off supernatant, every hole adds 150 μ L DMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing.By microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: inhibiting rate=(OD control group-OD treatment group)/OD control group * 100%.Result as shown in figure 13,0.01,0.05,0.1,0.2 and 0.4mg/mL G19 can suppress the growth of human pancreas cancer Panc-1 cell, inhibiting rate is respectively 27.41%, 33.27%, 61.28%, 92.89% and 98.31%, and has certain dose-dependently relation.
5, Grateloupia filicina (Wulf.) oligosaccharides G19 suppresses human lung adenocarcinoma A549 Growth of Cells
The good human lung adenocarcinoma A549 cell 100 μ L kinds of growth conditions of RPMI1640 substratum (the U.S. Hyclone company) culture medium culturing that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5v/v%CO
2incubator in cultivate after 24 hours and add respectively 100 μ L G19, make final concentration be respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, each concentration is established three multiple holes, continues to be placed in 37 ℃, 5%CO
2incubator in cultivate, after 48h, add 20 μ L5mg/mL MTT (U.S. Sigma company); Be placed in 37 ℃ of incubators and continue after reaction 4h, take out Tissue Culture Plate, sucking-off supernatant liquor, every hole adds 150 μ LDMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing, by microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: inhibiting rate=(OD control group-OD treatment group)/OD control group * 100%.Result as shown in figure 14,0.01,0.05,0.1,0.2 and 0.4mg/mL G19 can suppress the growth of human lung adenocarcinoma A549 cell, inhibiting rate is respectively 1.09%, 8.61%, 24.45%, 47.14% and 92.63%, and has certain dose-dependently relation.
6, Grateloupia filicina (Wulf.) oligosaccharides G19 does not affect the growth of Human normal hepatocyte LO2
The good Human normal hepatocyte LO2 cell 100 μ L kinds of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (the DMEM)/high glucose medium (U.S. Hyclone company) that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enter 96 orifice plates, every hole adds 4000 cells, is placed in 37 ℃, 5v/v%CO
2incubator in add respectively 100 μ L G19 after cultivating 24h, make final concentration be respectively 0.01,0.05,0.1,0.2 and 0.4mg/mL, each concentration is established three multiple holes, continues at 37 ℃, 5%CO
2incubator in cultivate, after 48h, add 20 μ L5mg/mL MTT (U.S. Sigma company).Be placed in 37 ℃ of incubators and continue after reaction 4h, take out Tissue Culture Plate, sucking-off supernatant, every hole adds 150 μ L DMSO (traditional Chinese medicines group), and concussion evenly, fully dissolves purple crystal thing, by microplate reader, detect, measure wavelength and be made as 490nm, measure the light absorption value (OD) in each hole, by following formula, calculate cell inhibitory rate: cell survival rate=OD treatment group/OD control group * 100%.As shown in figure 15, Grateloupia filicina (Wulf.) oligosaccharides G19 does not almost affect the growth of normal liver cell LO2 result in 0.01～0.4mg/mL dosage range.
7, the restraining effect of Grateloupia filicina (Wulf.) oligosaccharides G19 to transplanted tumor in nude mice growth
The preparation of 7.1 tested medicines
The tested medicine G19 preparing in the present invention is a Sulfated Polygalactan, soluble in water, and the physiological saline of take in experimentation is mixed with respective concentration solution, solution administration after 0.22 μ m filtering with microporous membrane as solvent.
The restraining effect experiment of 7.2 Grateloupia filicina (Wulf.) oligosaccharides G19 to neurospongioma U-87MG growth of xenografted in nude mouse
Purchased from the female Balb/c nude mouse right side oxter subcutaneous injection 200 μ L of Shanghai Slac Experimental Animal Co., Ltd., contain the cell suspension of 2 * 106 human glioma cell U-87MG (deriving from typical case's culture collection council of Chinese Academy of Sciences cell bank) 6 week age, vernier caliper measurement, treats that gross tumor volume grows to 100mm
3behind left and right, be divided at random negative control group and 25mg/kg G19 administration group, 6 of every group of nude mices, start administration.In administration group, every mouse gives the G19 (0.1mL/10g) that 2.5mg/mL physiological saline is prepared, fresh configuration before administration, and in control group, every mouse gives respective volume physiological saline.From tail intravenously administrable (using the fresh configuration of physiological saline administration), every two days once, continues medication 14 times, before each administration, claims mouse heavy.After administration, every 3 days with knurl volume of vernier caliper measurement, knurl volume V=a * b2/2, and wherein a is transplanted tumor longest distance, b is transplanted tumor shortest distance.Within the 26th day, put to death, it is 1244.0mm that the vernier callipers of take records the average knurl volume of control group
3, the average knurl volume of administration group is 521.2mm
3, calculating inhibiting rate=[(control group knurl volume-administration group knurl volume)/control group knurl volume] * 100%, inhibiting rate is 58.11%, result table 3 and as shown in figure 16, curve A is wherein control group, the G19 group that curve B is 25mg/kg.
The restraining effect (x ± s, n=6) of table 3 Grateloupia filicina (Wulf.) oligosaccharides G19 to transplanted tumor in nude mice growth
8, Grateloupia filicina (Wulf.) oligosaccharides G19 causes human glioma U-87MG cell-cycle arrest
The good human glioma U-87MG cell of the growth conditions of Dulbecco ' s Modified Eagle ' s Medium (DMEM)/high glucose medium (the U.S. Hyclone company) culture medium culturing that contains 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates enters six orifice plates with the density kind in 3 * 105/hole and spends the night.With 0.05mg/mL and 0.1mg/mL G19, process respectively after 24h, trysinization stops cultivating.1000 revs/min, 4 ℃ centrifugal 5 minutes, abandon supernatant, collecting cell.With 1mL precooling, PBS washes cell once, 1000 revs/min, 4 ℃ centrifugal 5 minutes, collecting cell.Add 100 μ L PBS re-suspended cells, slowly drip 300 μ L precooling dehydrated alcohols, put-20 ℃ of refrigerator fixed cells and spend the night.Before detection, 1000 revs/min, 4 ℃ centrifugal 5 minutes, remove stationary liquid with collecting cell.With 1mL PBS, wash cell twice, to eliminate residual ethanol.Add 450 μ L PBS re-suspended cell gently, and add 5 μ L RNase A (10mg/mL) (U.S. Thermo company).Put in 37 ℃, warm water bath and hatch 1 hour.After filtering with 300 order nylon membranes, adding final concentration is 10 μ L iodate pyridines (PI) (1mg/mL) (U.S. Sigma companies), and 4 ℃ of lucifuges are hatched 15 minutes.By flow cytometer (U.S. Becton Dickinson company), detect DNA content.Adopt ModiFit LT3.0 software (U.S. Becton Dickinson company) to carry out cell cycle analysis.Compare with blank group, 0.05mg/mL G19 processes after U-87MG cell 24h, and the cell in the S phase increases by 17.68%; And when the concentration of G19 increases to 0.1mg/mL, in S phase cell, increase by 59.96%.Result is as shown in table 4 and Figure 17, and Grateloupia filicina (Wulf.) oligosaccharides G19 can induce human glioma U-87MG cell block in the S phase, and presents certain dose-dependently relation.
Table 4 Grateloupia filicina (Wulf.) oligosaccharides G19 affected the U-87MG cell cycle
9, Grateloupia filicina (Wulf.) oligosaccharides G19 induction human glioma U-87MG apoptosis
The good human glioma U-87MG cell of growth conditions that Dulbecco ' s Modified Eagle ' s Medium (DMEM)/high glucose medium (U.S. Hyclone company) of containing 10%FBS (U.S. Gibco company), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates is cultivated enters six orifice plates with the density kind in 3 * 105/hole and spends the night.Add respectively 0.1 and 0.2mg/mL G19 process after cell 24h and 48h, with trysinization, stop cultivating.1000 revs/min, 4 ℃ centrifugal 5 minutes, abandon supernatant, collecting cell.With 1mL precooling, PBS washes cell once, 1000 revs/min, 4 ℃ centrifugal 5 minutes, collecting cell.Adopt
flour488Annexin V and PI (American I nvitrogen company) test kit is processed rear cell to G19 and is carried out apoptosis detection analysis.Configuration 1 * annexin-binding buffer adds 100 μ L1 * annexin-binding buffer in every pipe sample.Then add successively 2 μ L Alex Flour488Annexin V and 1 μ L PI (100 μ g/mL) working fluid.Set blank group simultaneously, singly dye group and two group of dying; The dyeing of room temperature lucifuge is after 15 minutes, and every pipe adds 400 μ L1 * annexin-binding buffer, mixes gently, is placed on ice; With 300 order nylon membranes, filter, flow cytometer carries out apoptosis analysis, adopts Cell Quest software (U.S. Becton Dickinson company) to carry out apoptosis and detects analysis.0.1mg/mL and 0.2mg/mL Grateloupia filicina (Wulf.) oligosaccharides G19 process after 24h, and apoptotic cell increases respectively 40.31% and 120.92%; When processing cell 48h, apoptotic cell increases by 125.13% and 490.02%.As shown in figure 18, oligosaccharides G19 can induce the remarkable apoptosis of human glioma U-87MG cell to result, and presents regular hour and dose-dependence.
By above experiment, can find out, Grateloupia filicina (Wulf.) oligosaccharides G19 provided by the invention in vitro and in body, have obvious antitumor action, and does not almost have toxicity to normal liver cell.In addition, this restraining effect is relevant to tumour cell cycle retardance and apoptosis of tumor cells.Therefore, Grateloupia filicina (Wulf.) oligosaccharides G19 is expected to be developed to antitumor drug as activeconstituents.
Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (7)
1. a Grateloupia filicina (Wulf.) oligosaccharides, is characterized in that: its molecular formula is C
48h
76o
52s
4na
4, there is following chemical structure:
2. a method of preparing Grateloupia filicina (Wulf.) oligosaccharides claimed in claim 1, is characterized in that: be first Grateloupia filicina (Wulf.) polysaccharide to be carried out to acidic hydrolysis, then carry out separation and purification.
3. method as claimed in claim 2, is characterized in that, comprises following concrete steps:
A) prepare Grateloupia filicina (Wulf.) polysaccharide;
B) Grateloupia filicina (Wulf.) polysaccharide step a) being obtained is first hydrolyzed at 60～100 ℃ with hydrochloric acid soln, and hydrolyzed solution, with in sodium hydroxide solution and rear concentrated, alcohol precipitation, centrifugal removal Grateloupia filicina (Wulf.) polysaccharide precipitation, is collected to supernatant liquor and is dried;
C) by step b) residue that obtains is soluble in water, carries out column chromatography, and water, 0.02～0.1mol/L NaCl solution and 1.5～2.5mol/L NaCl eluant solution, collect each several part elutriant successively;
D) by step c) the 0.05mol/L NaCl eluant solution that obtains part carries out column chromatography after desalination, then water, 0.01mol/L NaCl solution and 1.0～2.0mol/LNaCl solution carry out wash-out successively, collect elutriant, draw elution curve, press out peak sequencing 0.01mol/L NaCl eluant solution position is divided into A and B two portions;
E) by steps d) be dissolved in water after the A part elutriant that obtains is dry, then centrifuging and taking supernatant liquor carries out column chromatography, and with 0.15～0.2mol/L NaCl eluant solution, collection relative retention time is the component between 0.88～0.91;
F) by step e) gained relative retention time is 0.88～0.91 component is carried out column chromatography purification again, obtains described Grateloupia filicina (Wulf.) oligosaccharides, note by abridging as G19.
4. method as claimed in claim 3, is characterized in that: the concentration of hydrochloric acid soln step b) is 0.01～0.04mol/L, and the concentration of described sodium hydroxide solution is 1～2mol/L.
5. an application for Grateloupia filicina (Wulf.) oligosaccharides claimed in claim 1, is characterized in that: using described Grateloupia filicina (Wulf.) oligosaccharides as activeconstituents for the preparation of antitumor drug.
6. application as claimed in claim 5, is characterized in that: using described Grateloupia filicina (Wulf.) oligosaccharides as unique activeconstituents for the preparation of antitumor drug.
7. the application as described in claim 5 or 6, is characterized in that: described tumour comprises at least one in neurospongioma, liver cancer, carcinoma of the pancreas, adenocarcinoma of lung.
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| CN101077356A (en) * | 2006-05-24 | 2007-11-28 | 上海中医药大学 | Application for polysaccharide extraction of grateloupia filicina in preparing antitumor medicine and other medicines |
| CN101851298A (en) * | 2010-05-17 | 2010-10-06 | 上海和臣医药工程有限公司 | Sulfated galactan and preparation method thereof |
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| CN101077356A (en) * | 2006-05-24 | 2007-11-28 | 上海中医药大学 | Application for polysaccharide extraction of grateloupia filicina in preparing antitumor medicine and other medicines |
| CN101851298A (en) * | 2010-05-17 | 2010-10-06 | 上海和臣医药工程有限公司 | Sulfated galactan and preparation method thereof |
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