CN106349397B - Depolymerization glycosaminoglycan extracted from sea cucumber composition and the preparation method and application thereof - Google Patents
Depolymerization glycosaminoglycan extracted from sea cucumber composition and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of depolymerization glycosaminoglycan extracted from sea cucumber composition and its preparation method and application, it is one or more in the depolymerization glycosaminoglycan extracted from sea cucumber of 2000Da to 12000Da for weight average molecular weight, and the preparation method of depolymerization glycosaminoglycan extracted from sea cucumber composition includes extraction, purifying glycosaminoglycan extracted from sea cucumber and degradation glycosaminoglycan extracted from sea cucumber.The present invention is screened by many experiments, and using the sea cucumber of Echinodermata as research object, the extraction and purification process of glycosaminoglycan extracted from sea cucumber and the degradation technique of depolymerization glycosaminoglycan extracted from sea cucumber are preferably gone out by many experiments.Entire technological design is reasonable, and operability is strong, it can be achieved that industrialized production.It is antitumor research shows that, the tube chamber that depolymerization glycosaminoglycan extracted from sea cucumber composition can significantly inhibit huve cell in vitro generates, it can inhibit the transfer of melanoma and breast cancer in vivo, with good antitumous effect, it is expected to be developed into antitumor drug or health products efficient, that toxic side effect is small, it is with important application prospects.
Description
Technical field
The invention belongs to medicine and health product technology fields, are 2000Da- the present invention relates to weight average molecular weight range
Depolymerization glycosaminoglycan extracted from sea cucumber composition of 12000Da and preparation method thereof and the depolymerization glycosaminoglycan extracted from sea cucumber composition are pre-
Application in anti-and treatment tumor disease.
Background technology
Malignant tumour is a kind of common disease, seriously threatens the life of the mankind.Current main treatment means are operations, put
Treatment, chemotherapy and immunization therapy.The chemotherapy of one for the treatment of means, there are two big obstacles while inhibiting, killing tumour cell is
Its toxic side effect and drug resistance.Toxic side effect is embodied in not only killing tumor cell, but also kills normal tissue cell, to human body
It causes toxicity, reduce patient's physiological function.Therefore, research and development can have attenuation, increase with antitumor drug conjunctive use
Quick, synergy newtype drug has important positive effect.
Glycosaminoglycan extracted from sea cucumber (Holothurian Glycosaminoglycan, abbreviation HG or HGAG), structural formula is such as
Under, it is a kind of sulfated glycosaminoglycan of the branch containing fucose extracted from sea cucumber, structure is approximate with heparin (Heparin) but
There were significant differences again.HG composition monosaccharide includes amine-galactose, glucuronic acid, fucose and their sulfate group,
Middle glucuronic acid and amine-galactose are interconnected by β (1-3) and β (1-4) glycosidic bond, formed in the mammalian body
The main chain of the similar disaccharides constitutional repeating units of chondroitin sulfate E, and fucose and its sulfate group are connected in the form of side chain
Main chain (Vieira RP et al., J.Biol.Chem., 1991,266:13530-13536).In addition, different sea cucumber sources and
HG obtained by preparation method in structure there is also different degrees of difference (Ken-ichiro Y et al.,
Tetrahedron Letters,1992,33:4959), be mainly manifested in amine-galactose in sugar backbone the positions 4- and 6- sulphur
The difference of Esterification difference and the Sulfation on fucose side chain.
Inside and outside research confirms that HG has good anticoagulation, anti thrombotic action.Though HG belongs to glycosaminoglycan with heparin
Family, and biological activity is also extremely similar, but HG may act on multiple links of anti-freezing, cause bleeding it is dangerous compared with
It is small.
It is well known that heparin is that anticoagulating active is strongest in the natural products found at present, and it is early have been observed that its
Anti-inflammatory and anti-tumor aspect has good bioactivity, and heparin is independent or is used in combination with other drugs, is proved various
In pre-clinical tumor models and clinical treating malignant tumor effectively.Some low molecular weight heparin, such as dalteparinSodium (Dalteparin)
It has been widely used in antitumor clinical treatment, has mainly taken the mode with antitumor drug conjunctive use, but since it is anti-
Solidifying activity, FDA applications of the official approval dalteparinSodium in terms of oncotherapy not yet.Because of the anticoagulation of heparin class, clinical
Tumour can be treated using its anti-freezing dosage, effect is limited, so if retaining antitumor work while reducing anticoagulating active
Property can increase Clinical practice dosage, to greatly enhance antitumous effect, while significantly reduce bleeding risk.
Glycosaminoglycan extracted from sea cucumber is significantly reduced relative to heparin, anticoagulating active.The present inventor, which tests, to be found, depolymerization sea cucumber osamine
The anticoagulating active of glycan (dHG) has an apparent reduction again compared to the glycosaminoglycan of non-depolymerization, dHG may have and avoid (no matter
It is unassorted macromolecular heparin or low molecular weight heparin) heparin-induced platelet aggregation reduces disease (Hepaein-Induced
Thrombocytopenia, HIT) fatal risk.Therefore, if can the smaller dHG of adverse reaction be applied to clinical tumor
Treatment will bring glad tidings for tumor patient.At home and abroad there is no dHG to be applied to antitumor report at present.
Invention content
Goal of the invention：The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of curative for effect, safety
Good weight average molecular weight is the depolymerization glycosaminoglycan extracted from sea cucumber composition of 2000Da-12000Da；Another object of the present invention is to provide
The preparation method that depolymerization glycosaminoglycan extracted from sea cucumber composition is simple and efficient, the present invention is to the depolymerization glycosaminoglycan extracted from sea cucumber group that is prepared
The antitumor experimental study in inside and outside that object has carried out system is closed, finds it with good antitumor activity, therefore can be used for
Prepare antitumor drug or health products.
In order to achieve the goal above, the technical solution that the present invention takes is：
A kind of depolymerization glycosaminoglycan extracted from sea cucumber composition, it is weight average molecular weight between the depolymerization sea cucumber of 2000Da-12000Da
Glycosaminoglycan it is one or more.
The preparation method of depolymerization glycosaminoglycan extracted from sea cucumber composition provided by the invention comprising following steps：
1) it extracted from sea cucumber, purify glycosaminoglycan extracted from sea cucumber；
2) glycosaminoglycan extracted from sea cucumber that degradation extracts.
Preferably, the preparation method of above-described depolymerization glycosaminoglycan extracted from sea cucumber composition, in the step 1)
Sea cucumber selected from but not limited to the sea of sand ginseng of following Echinodermata, Holothuria atra, hojothuria leucospilota, Holothuria scabra, Holothuria nobilis Selenka, lattice skin
It is one or more in family name sea cucumber, actinopyga etc..Wherein hojothuria leucospilota originates in China's South China Sea, is distributed wide, stock number
Greatly, cheap and sugar content is high, is optimal raw material.As other schemes, glycosaminoglycan of the present invention can
With one or more in Echinodermata.
The preparation method of the depolymerization glycosaminoglycan extracted from sea cucumber composition, which is characterized in that step 1) is extracted from sea cucumber
The step of glycosaminoglycan extracted from sea cucumber includes that separation is rubbed, digested and precipitated to it to obtain glycosaminoglycan extracted from sea cucumber crude product；The enzyme
Solution enzyme used includes one or more in alkali protease, neutral proteinase, pancreatin, papain；The precipitation packet
It includes and is precipitated with organic solvent methanol, ethyl alcohol, isopropanol or acetone, separation includes ion exchange resin separation and metal salinity
Grade precipitation.
Preferably, the preparation method of above-described depolymerization glycosaminoglycan extracted from sea cucumber composition, step 1) purifying sea
The step of ginseng glycosaminoglycan includes that spent ion exchange resin is adsorbed and be classified elution, hydrogen peroxide for decoloration and except heat source obtains sea
Ginseng glycosaminoglycan sterling；
Ion exchange resin used is cation exchange resin or anion exchange resin；The elution refers to using water
Classification elution is carried out with the inorganic salt solution of a concentration of 1~20% (m/m).
Preferably, the preparation method of above-described depolymerization glycosaminoglycan extracted from sea cucumber composition, step 2) degradation carry
The step of glycosaminoglycan extracted from sea cucumber of taking-up includes：In acidic environment, temperature is controlled between 20-80 DEG C, is degraded using hydrogen peroxide
Reaction 2-48 hours, degradation obtain depolymerization glycosaminoglycan extracted from sea cucumber composition of the weight average molecular weight between 2000Da-12000Da；
The dioxygen water consumption is between 0.1-30% (v/v)；Acid used in the acidic environment be formic acid, acetic acid,
Propionic acid, hydrochloric acid or sulfuric acid；The dosage of the acid is between 0.1-20% (v/v).
Preferably, the preparation method of above-described depolymerization glycosaminoglycan extracted from sea cucumber composition, step 2) degradation carry
The glycosaminoglycan extracted from sea cucumber of taking-up includes the sodium chloride fractional precipitation desalination with various concentration, is then freeze-dried or is dried under reduced pressure,
Obtain the depolymerization glycosaminoglycan extracted from sea cucumber composition that dispersion degree is less than 1.5.
A kind of Halth-care composition, including depolymerization glycosaminoglycan extracted from sea cucumber composition and the acceptable carrier of health products trade.
A kind of pharmaceutical composition, including depolymerization glycosaminoglycan extracted from sea cucumber composition of the present invention and pharmaceutically acceptable
Carrier.
Depolymerization glycosaminoglycan extracted from sea cucumber composition of the present invention is preparing prevention and treatment tumor disease drug or health care
Application in product.
Depolymerization glycosaminoglycan extracted from sea cucumber composition of the present invention is preparing prevention and treatment tumor disease drug or health care
Application in product, be used alone or with antitumor drug use in conjunction.
The present invention optimizes to obtain the extraction and purification process of glycosaminoglycan extracted from sea cucumber by many experiments, improves glycosaminoglycan extracted from sea cucumber
Biodegrading process, entire preparation process reasonable design, operability is strong, and the depolymerization glycosaminoglycan extracted from sea cucumber composition to obtaining into
Row structural characterization obtains depolymerization glycosaminoglycan extracted from sea cucumber composition of the weight average molecular weight between 2000Da and 12000Da.By this hair
Depolymerization glycosaminoglycan extracted from sea cucumber composition prepared by bright the method, molecular weight distribution, optical activity, sulfate group composition ratio,
Means known in the art detection can be used in the quality control indexs such as nuclear-magnetism.
The present invention has carried out the antitumor research in inside and outside to the depolymerization glycosaminoglycan extracted from sea cucumber composition.Gained of the invention
To depolymerization glycosaminoglycan extracted from sea cucumber dHG can significantly inhibit in vitro huve cell (HUVEC) tube chamber generate, in vivo
The lung of melanoma and breast cancer can be inhibited to shift, and have apparent dose-effect relationship, there is good antitumous effect.
Description of the drawings
Fig. 1 is the molecular weight distribution of depolymerization glycosaminoglycan extracted from sea cucumber dHG.
Fig. 2 is glycosaminoglycan extracted from sea cucumber HG's and depolymerization glycosaminoglycan extracted from sea cucumber dHG1H-NMR。
Fig. 3 is HUVEC segment dislocation results.
Fig. 4 is that Pulmonary artery stove counts block diagram in breast cancer model.
Fig. 5 is Pulmonary artery coverage rate block diagram in breast cancer model.
Specific implementation mode
The present invention is further described by the following embodiment, but do not limit the invention in any way.
Embodiment 1：The extraction and purifying of glycosaminoglycan extracted from sea cucumber (HG)
1. weighing dry hojothuria leucospilota medicinal material 5Kg, water soaked overnight.It cleans, takes body wall, drain away the water, rub, weigh simultaneously
Moisturizing is to 25Kg.60 DEG C of water-baths are set, it is 8.0 to adjust pH with sodium hydroxide, and 2% alkali protease2709 is added, and is digested 6 hours
Afterwards, the pancreatin for adding 2% continues enzymolysis 16 hours, notices that control pH is alkalescent in enzymolysis process.It is centrifuged after enzymolysis,
Isometric ethyl alcohol alcohol precipitation is added in supernatant, is collected by centrifugation solid, then with the water dissolution of 30 times of quality, and it is solid that 1%NaCl is added
Body is allowed to whole dissolvings, and 1Kg (weight in wet base) 1074 type ion exchange resin is then added, and adjusts pH to 8.0, suction is stirred at room temperature
It attached 4 hours, after the completion of absorption, filters, filtered resin is first washed with clear water, then respectively with 7% (w/v) and 10%
(w/v) respectively elution 3 times, every time 15 minutes of NaCl solution.The eluent for merging 10%NaCl is collected, and 0.8 times of volume is added
Ethyl alcohol alcohol precipitation, stood overnight under the conditions of 4 DEG C, then collected by suction precipitate and crude product sea cucumber osamine is dried under reduced pressure to obtain at 45 DEG C
Glycan 24g.
2. the water dissolution of above-mentioned 20 times of quality of glycosaminoglycan extracted from sea cucumber crude product of gained, adjusting pH with 4M NaOH solutions is
10, hydrogen peroxide, which is then added, makes its a concentration of 3% (v/v) carry out decolorization, and reaction 2 hours is stirred at room temperature.After reaction
1%NaCl and isometric ethyl alcohol alcohol precipitation is added, obtains the water dissolution that solid uses 10 times of amounts again, then with 1%NaCl and ethyl alcohol alcohol precipitation 2
It is secondary.The solid of gained is dried under reduced pressure after washing 2 times with ethyl alcohol in 45 DEG C, obtains sterling glycosaminoglycan extracted from sea cucumber 22.5g.
Embodiment 2：The preparation of depolymerization glycosaminoglycan extracted from sea cucumber (dHG)
The preparation of 1.dHG
Sterling glycosaminoglycan extracted from sea cucumber 20g obtained described in Example 1 is added 160mL water dissolutions, and 19.0mL vinegar is added
Acid and 60.0mL hydrogen peroxide cool down after being stirred to react 22 hours at 60 DEG C in ice bath, and 4M NaOH solutions are then added and adjust
PH to 9.5 adds the ethyl alcohol alcohol precipitation of 2 times of volumes, stands, and centrifuges to obtain precipitation, as depolymerization glycosaminoglycan extracted from sea cucumber crude product.
It takes the crude product again with 200mL water dissolutions and 3%NaCl solids is added, with 1 times of volume ethanol alcohol precipitation, gained precipitation is multiple
Again respectively with 2%, 1%NaCl solids and 1 times of volume ethanol alcohol precipitation 2 times after molten, finally obtained precipitation is washed 2 times with ethyl alcohol,
Then it is dried under reduced pressure to obtain 18.0g depolymerization glycosaminoglycan extracted from sea cucumber (dHG).
The physicochemical property of 2.dHG
2.1 molecular weight distribution
Gained depolymerization glycosaminoglycan extracted from sea cucumber (dHG) is analyzed with HPGPC, and chromatographic column is a TSKgel G2000SW (7.8mm
× 30cm, 5 μm, product identification：T08542) and TSKgel G3000SW (7.8mm × 30cm, 5 μm, product identification：
T10407-02T) connection uses, and it is 10092Da, dispersion degree 1.3 to measure weight average molecular weight.(40.5min in figure as shown in Figure 1
Place is solvent peak).
2.2 optical activity：It is measured according to two annex VIE methods of Chinese Pharmacopoeia (2010 editions).Use WZZ-1S type automatic rotaries
Light instrument, sodium lamp (λ589nm), sample cell 1dm.Measurement result is -56.5 °, with sterling glycosaminoglycan -60.5 ° of phases of optical activity
Closely.The result shows that the degradation technique that the present invention uses will not change its steric configuration.
2.3 sulfate radical carboxylate radical ratios：Conductometric Titration Method, measure dHG sulfate radical carboxylate radical ratio be 3.84 with
Sterling glycosaminoglycan 3.55 is consistent.The result shows that the degradation technique that the present invention uses will not reduce sulfate radical content.
2.4 1H-NMR
Glycosaminoglycan extracted from sea cucumber HG's that the present invention is prepared and depolymerization glycosaminoglycan extracted from sea cucumber dHG1H-NMR collection of illustrative plates such as Fig. 2
It is shown, the collection of illustrative plates show to degrade front and back glycosaminoglycan extracted from sea cucumber signal characteristic it is almost the same, only the low signal of molecular weight is more
Clearly, also indicate that significant changes do not occur for the front and back basic chemical structure of glycosaminoglycan extracted from sea cucumber degradation.And δ 1.33ppm are rock algae
Methyl characteristic peak and δ 1.92ppm on glycosyl are the acetyl group characteristic peak on amine-galactose, show glycosaminoglycan extracted from sea cucumber
Monosaccharide forms the common trait for including amine-galactose and fucose side chain.And the main difference of different glycosaminoglycan extracted from sea cucumber
It is the difference of the positions 4- of amine-galactose in main chain and the difference of 6- Sulfations and the Sulfation on fucose side chain
It is different.According to atlas analysis, there is apparent characteristic peak in δ 4.22ppm and δ 4.8ppm or so illustrate hojothuria leucospilota without characteristic peak
There is only 6- Sulfations and without 4- Sulfations on the amine-galactose of glycosaminoglycan, (Fan paints once etc., medicine with document for this
Learn journal, 1983,18 (3)：203) report is consistent.And on side chain fucosido, there is the 2 of about 15%, 4- di-sulfates
Base (δ 5.65ppm characteristic peaks), about 30% 3,4- di-sulfates base (δ 5.26ppm and 4.90ppm characteristic peak) and about 20%
4- sulfate groups (δ 5.34ppm and 5.35ppm characteristic peak).In addition to the characteristic peak having been reported that above, there is also such as δ at low field
Two characteristic peaks of 5.10ppm and δ 5.00ppm, and chemical displacement value is more slightly lower than the characteristic peak of above-mentioned report, illustrates the sea cucumber
There are the sulfate group of 2- or 3- are monosubstituted for glycosaminoglycan.
2.5 anticoagulating actives detect
The present invention determines the depolymerization glycosaminoglycan extracted from sea cucumber dHG that 2 gained weight average molecular weight of embodiment is 10092Da and reaches liver
The anti-xa activity of element.It the results are shown in Table 1：
1 anticoagulating active of table detects
As a result the external anticoagulating active of prompt depolymerization glycosaminoglycan extracted from sea cucumber dHG only has 1 the percent of dalteparinSodium, illustrates to solve
Poly- glycosaminoglycan extracted from sea cucumber dHG bleeding risk smallers, safety higher, the setting for Subsequent pharmacological dosage provide foundation.
Embodiment 3：Depolymerization glycosaminoglycan extracted from sea cucumber dHG acute toxicity testings
1. experiment material
DHG10092 (prepares gained) by 2 method of the embodiment of the present invention；ICR mouse.
2. experimental method
ICR mouse are randomly divided into 2 groups (every group 10, half male and half female) according to weight, and tail vein gives dosage and is respectively
The dHG10092 of 4000mg/kg and 2000mg/kg.
3. experimental result
4000mg/kg dosage groups Some Animals tail vein administration after, have a convulsion, have difficulty in breathing immediately, abdominal respiration,
Phenomena such as righting reflex loss and the urinary incontinence, above-mentioned symptom is alleviated after administration 3 minutes, but spontaneous activity is apparent compared with the control group
It reduces；
2000mg/kg dosage groups animal does not occur apparent abnormal symptom after tail vein administration.Each group animal body after administration
Weight decline, wherein 4000mg/kg groups administration 1 day after the weight of animals there are significant difference (P compared with solvent control group<
0.05)；The weight of animals restores to increase from the 2nd day after administration, and that there are no significant compared with solvent control group is poor for each group the weight of animals
It is different.Each group is showed no animal dead in observation period, apparent abnormal without other.Terminate observation, CO within the 14th day after administration2Put to death animal
After be dissected and observed, each group animal main organs are visible by naked eyes exception.The experimental results showed that dHG provided by the invention
10092 safeties are preferable, still safe when dosage reaches 4000mg/kg.
Embodiment 4：Depolymerization glycosaminoglycan extracted from sea cucumber dHG studies the influence that HUVEC cell luminal structures are formed
1. experiment material
Test-compound：The weight average molecular weight that embodiment 2 is prepared is the depolymerization glycosaminoglycan extracted from sea cucumber of 10092Da
(dHG10092), card is rich replaces Buddhist nun (Cabozantinib).
Cell strain：Primary HUVEC cell lines are purchased from Australia Sai Ersi (Allcells) company.
Reagent：Endothelial basal medium (Australia Sai Ersi, Cat#HUVEC-004B), endothelial cell complete medium
(Australia Sai Ersi, Cat#HUVEC-004), Geltrex LDEV-Free Reduced Growth Factor Basement
Membrane Matrix (without phonol red) (GIBCO, cat#A1413202), 0.25%Trypsin-EDTA
(GIBCO, Cat#25200), fetal calf serum (GIBCO, Cat#10099141), mould strepto- dual anti-solution (GIBCO, Cat#
15140-122), dimethyl sulfoxide (DMSO) (Sigma, Cat#D2650).
2. experimental method
2.1 test-compounds are prepared：Card is rich to be dissolved in DMSO for Buddhist nun and is made into 20mM storing liquids.In DMSO gradient dilution at
The storing liquid of 200 times of final concentrations.The depolymerization glycosaminoglycan extracted from sea cucumber dHG10092 that the present invention is prepared is dissolved in 0.9%NaCl and is made into
20mM storing liquids.Use 0.9%NaCl gradient dilutions at the storing liquid of 50 times of final concentrations again.(contained with basal medium again when dosing
2%FBS) it is diluted to the compound solution of 2 times of final concentrations.
2.2 segment dislocation experimental methods：Add 50 holes μ L/ Geltrex glue to 96 orifice plates, is then placed in 37 DEG C of incubators and is incubated
1 hour, gelling is made to consolidate.Take HUVEC cells (the complete medium culture of cell, experiment the previous day of cell coverage rate about 80%
With the basal medium overnight starvation containing 2%FBS), cell is collected by centrifugation with pancreatin digestion, with basal medium (containing 2%FBS)
It is resuspended and counts.Cell suspension is diluted to 2 × 10 with basal medium (containing 2%FBS)5A cell/ml, then hangs cell
The compound solution 1 of liquid and 2 times of final concentrations:1 mixing.Cell suspensions of the 100 μ L containing 1 × compound is uniformly spread to coating
In 96 orifice plates of Geltrex glue (1 × 104/hole), multiple holes detection.It takes pictures under the microscope after 4 hours, and counts formation
Tube chamber number.With CTG method detection compound toxicity.GraphPad Prism software development dose-effect figures.
3. result
The experimental results showed that the dHG10092 that is prepared of the present invention shows Human Umbilical Vein Endothelial Cells in 50 μM of concentration
HUVEC segment dislocations have more significant inhibiting effect, and have dose-dependence.Segment dislocation data statistics result is shown in respectively
Fig. 3.
Embodiment 5：The influence that depolymerization glycosaminoglycan extracted from sea cucumber dHG shifts B16F10 melanoma cells spontaneous lungs
1. experiment material
1.1 experimental animals and cell
Cleaning grade C57BL/6 mouse (female, 6-8 week old, 16-20g), the limited public affairs of tonneau China's experimental animal technology are tieed up in Beijing
Department provides.
1.2 experimental drug
DHG10092 (prepares gained) by 2 method of the embodiment of the present invention；DalteparinSodium.
1.3 experiment reagent
DMEM (GiBCO companies of the U.S.), phosphate buffer (PBS), calf serum, trypsase (Trypsin), second two
Amine tetraacethyl (EDTA), formaldehyde, physiological saline, sodium bicarbonate.
1.4 experiment equipment
Lycra inverted fluorescence microscope, accurate pipettor, full-automatic high-pressure autoclave, superclean bench, ultra low temperature freezer,
CO2Incubator, pure water meter, electronic balance, table electrothermal air dry oven, refrigerator, liquid nitrogen container, centrifuge, pH meter, syringe.
2. experimental method
2.1 prepare cell suspension
Routine culture B16F10 tumour cells, 4-5 days before experiment, by 1:10, cell passes in Tissue Culture Flask, in order to avoid
Not exclusively converging excessively occurs in cell growth.Each culture bottle is greatly containing about 6-8 × 106Cell, the cell of logarithmic growth phase, is abandoned
Culture solution is removed, is cleaned with PBS, 0.25% pancreatin -0.02%EDTA digestive juices of 1mL are added, are put into cell incubator, 1-
After 3min, tapping culture bottle makes cell detachment.10mL DMEM are added, blows and beats cell with pipette, obtains single cell suspension, turn
Enter 50mL polypropylene centrifuge tubes.Centrifuge cell (1200rpm × 10min), is then washed twice with PBS.Cell is counted with trypan blue,
It is 2.5 × 10 to adjust cell concentration6A/mL.
2.2 replicate B16F10 experimental tumor metastasis models
C57BL/6 mouse are taken to be placed in mouse injection fixator, tail vein injection 0.2mL cell suspensions (contain 2 × 105It is swollen
Oncocyte).The mouse of inoculated tumour cell is randomly divided into three groups：Model control group (physiological saline), administration group：System of the present invention
Standby obtained dHG10092 dosage groups (20mg/kg), dalteparinSodium group (20mg/kg) are subcutaneously injected drug, are anaesthetized after 20 days, locate
Dead mouse, takes lungs.It avoids tweezers from touching organ surface, prevents injury tissue, tissue is cleaned with PBS.10%PBS- formals
Woods fixes lung tissue, weighs lung weight, takes pictures, influence of the evaluation drug to B16F10 blood Lung metastases.
3. experimental result
3.1 weight
In experimentation, mouse weight is without significantly reducing
3.2 lung weights
There were significant differences for the lung of administration group and physiological saline group weight.DalteparinSodium group significantly inhibits lung and increases again, and the present invention carries
The dHG10092 of confession also inhibits the growth of lung weight.The inhibiting rate that the inhibiting rate of dalteparinSodium reaches 83.6%, dHG10092 reaches
55.7%.
3.3 lung tumors tubercle numbers
Compared with control group (physiological saline group), the tumor nodule number of dalteparinSodium group and dHG10092 groups significantly reduces.It is right
It is respectively 99,17,34 according to group (physiological saline group), dalteparinSodium group and dHG10092 group lung tumors tubercle numbers.
The experimental results showed that, the depolymerization glycosaminoglycan extracted from sea cucumber dHG10092 that the present invention is prepared is in 20mg/kg agent above
Amount, which is used alone, can significantly inhibit metastases, and not influenced on mouse weight.
Embodiment 6：The influence that depolymerization glycosaminoglycan extracted from sea cucumber dHG shifts 4T1 mouse mastopathy cell spontaneous lungs
1. experiment material
1.1 experimental animals and cell
Cleaning grade BALB/C mice (female, 6-8 week old, 16-20g), the limited public affairs of tonneau China's experimental animal technology are tieed up in Beijing
Department provides.
1.2 experimental drug：
DHG10092 (prepares gained) by 2 method of the embodiment of the present invention；Cis-platinum (cisplatin)；DalteparinSodium.
1.3 experiment reagent：
DMEM (GiBCO companies of the U.S.), phosphate buffer (PBS), calf serum, trypsase (Trypsin), second two
Amine tetraacethyl (EDTA), formaldehyde, physiological saline, sodium bicarbonate.
1.4 experiment equipment
Lycra inverted fluorescence microscope, accurate pipettor, full-automatic high-pressure autoclave, superclean bench, ultra low temperature freezer,
CO2Incubator, pure water meter, electronic balance, table electrothermal air dry oven, refrigerator, liquid nitrogen container, centrifuge, pH meter, syringe.
2. experimental method
2.1 cell culture
4T1 cells use RPMI-1640 culture solution cultures, wherein a concentration of the 10% of the fetal calf serum added, penicillin/
Streptomysin is dual anti-to press 1：:100 dilution proportion is incubated in culture solution containing 5%CO2Saturated humidity is 37 DEG C of constant incubators
In.Every 2 to 3 days of cell is with 1:3-1:5 passages.
When 2.2 excision primary tumor the case where mouse Pulmonary metastasis focuses
BALB/C mice 70 is randomly divided into 7 groups, the 4th fat pad of mammary gland inoculation 1 × 1054T1 cells.Each subsequent set of point
It is 20mg/kg, 40mg/kg, 80mg/kg, dalteparinSodium 4mg/kg not give 10092 dosage of dHG.After successive administration 12 days, excision
Mammary gland region tumors, at the 13rd day, every group of increase cis-platinum 4mg/kg, once every four days.At the 30th day, anesthesia was put to death mouse, is taken
Go out lungs and spleen, clean, weigh, 10%PBS- formalin fixes lung tissue, influence of the evaluation drug to Lung metastases.
3. experimental result
3.1 weight
All mouse (alone or in combination) weight after plus cisplatin in treatment have decline, are shown to be cis-platinum and result in weight
Decline.
3.2 knurl weight
The tumour growth of primary tumor is not significantly different between different groups, but cis-platinum and dHG10092 drug combination groups
Compared with individual cis-platinum group, knurl weight is reduced.
3.3 lungs weight and spleen weight
Group (dHG10092 that cisplatin alone or cis-platinum are prepared with the present invention is shared) after plus cisplatin in treatment, with
Negative control group is compared, and lung weight and spleen significantly reduce again, show treatment group and can inhibit metastases and with the relevant bone of tumour
Myelocytic diffusion.
The transfer quantization of 3.4 lungs
Compared with cis-platinum group is used alone, dHG 10092 provided by the invention can obviously reduce lung with cisplatin combined medication
Portion's tumor nodule number, and be in dose-dependence, 80mg/kg groups are considerably less than independent group of (P of cis-platinum<0.05), as shown in Figure 4.
HE is dyed, quantization lung tumors region, as a result prompts all plus cisplatin in treatment groups (cisplatin alone or cis-platinum and this hair
The bright dHG10092 being prepared is shared) lung tumors regional extent (p can be significantly reduced<0.0001), and the present invention carries
The dHG 10092 of confession shows that dose-effect relationship, 80mg/kg dosage group curative effects are best.As shown in Figure 5.
The experimental results showed that, depolymerization glycosaminoglycan extracted from sea cucumber dHG 10092 and Cisplatin provided by the invention can be shown above
It writes and inhibits metastases, and dose-dependence, 80mg/kg dosage group most pronounced effects are presented, and do not find any bad anti-
It answers.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of depolymerization glycosaminoglycan extracted from sea cucumber composition, which is characterized in that it for weight average molecular weight between 2000Da extremely
The depolymerization glycosaminoglycan extracted from sea cucumber of 12000Da it is one or more.
2. the preparation method of the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 1, which is characterized in that including following step
Suddenly：
1) it extracted from sea cucumber, purify glycosaminoglycan extracted from sea cucumber；
2) glycosaminoglycan extracted from sea cucumber that degradation extracts；
Step 2) degrade extract glycosaminoglycan extracted from sea cucumber the step of include：In acidic environment, temperature is controlled at 20-80 DEG C
Between, using hydrogen peroxide degradation reaction 2-48 hours, degradation obtained the depolymerization sea cucumber suger that weight average molecular weight is less than 12000Da
Amine glycan pool object；
The dioxygen water consumption is between 0.1-30%；Acid used in the acidic environment is formic acid, acetic acid, propionic acid, salt
Acid or sulfuric acid；The dosage of the acid is between 0.1-20%.
3. according to the preparation method of the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 2, which is characterized in that the step
It is rapid 1) in sea cucumber include the sea of sand ginseng of Echinodermata, Holothuria atra, hojothuria leucospilota, Holothuria scabra, Holothuria nobilis Selenka, the seas lattice Pi Shi
It is one or more in ginseng, actinopyga.
4. according to the preparation method of the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 2, which is characterized in that step
1) the step of glycosaminoglycan extracted from sea cucumber is extracted from sea cucumber include it is rubbed, is digested, fractional precipitation, separation obtain sea cucumber suger
Amine glycan crude product；Enzyme used in the enzymolysis includes one kind in alkali protease, neutral proteinase, pancreatin, papain
Or it is a variety of；The precipitation includes being precipitated with organic solvent methanol, ethyl alcohol, isopropanol or acetone, and separation includes ion exchange
Resin detaches and metal salt fractional precipitation.
5. according to the preparation method of the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 2, which is characterized in that step
1) the step of purifying glycosaminoglycan extracted from sea cucumber includes that spent ion exchange resin is adsorbed and be classified elution, and hydrogen peroxide for decoloration simultaneously removes heat
Source obtains glycosaminoglycan extracted from sea cucumber sterling；
Ion exchange resin used is cation exchange resin or anion exchange resin；The elution refer to water and
The inorganic salt solution that mass concentration is 1~20% carries out classification elution.
6. the preparation method of depolymerization glycosaminoglycan extracted from sea cucumber composition according to claim 2, which is characterized in that step 2)
The glycosaminoglycan extracted from sea cucumber extracted of degrading includes then being freeze-dried or being subtracted come desalination with the sodium chloride fractional precipitation of various concentration
Press dry it is dry, obtain dispersion degree be less than 1.5 depolymerization glycosaminoglycan extracted from sea cucumber composition.
7. a kind of Halth-care composition, which is characterized in that including the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 1
With the acceptable carrier of health products trade.
8. a kind of pharmaceutical composition, which is characterized in that including described in claim 1 depolymerization glycosaminoglycan extracted from sea cucumber composition and
Pharmaceutically acceptable carrier.
9. the depolymerization glycosaminoglycan extracted from sea cucumber composition described in claim 1 is preparing prevention and treatment tumor disease drug or guarantor
Application in strong product.
10. depolymerization glycosaminoglycan extracted from sea cucumber composition according to claim 9 is preparing prevention and treatment tumor disease drug
Or the application in health products, it is characterised in that be used alone or with antitumor drug use in conjunction.
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