CN1583803A - Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity - Google Patents
Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity Download PDFInfo
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Abstract
Glycan ingredient MF4 has increased immunological competence and anti-tumour functions, which is separated from sea-animal thick shell mussel. Its relative molecular weight is 113,000. It was analyzed and identified the physical properties and chemical structure through multiple spectra and methods, of which spectra are such as UV, IR, GC/MS,1HNMR, of which chemical methods are such as hydrolysis, acetylization, periodate oxidation, Smith degradation, methylation, sulfuric acid- carbazole reaction. Testing in vitro proves that MF4 promotes T,B lymph cells proliferation. Anti-tumour testing in vivo shows that small rat with subcutaneous inoculation is capable of inhibiting Lewis lung cancer and S-180 caruncle's growth, and has promotion functions for NK cell activity and lymph cell transfer of charged Lewis lung cance small rat. The thick shell mussel glycan MF4 is used to prepare new immonological strengthen pharmaceutics and anti-tumour drugs. And it's a new approach for developing Chinese sea biological sources.
Description
Technical field
The present invention relates to medical technical field, is to be separated to a kind of new polysaccharide MF4 with enhancing immunity and anti-tumor activity from the marine animal Mytilus crassitesta Lischke.
Background technology
Mytilus crassitesta Lischke (Mytilus Coruscus) belongs to Mytilidae (Mytilidae) mussel and belongs to (Mytilus); Be the warm water kind, mainly be distributed in the Dalian in Liaoning Province, little Chang Mount in China; The Qingdao of Shandong Province, Tuo Jidao, big Qin Dao, Peng Lai, Yantai, Weihai; The Shengsi archipelago in Zhejiang Province, Zhoushan Islands, Zhu family's point, fish mountain group island, following Da Chen, Nan Jidao; Flat Tan in Fujian Province, Xiamen, Dongshan.Shell is bigger, and is thick and heavy, is wedge shape; About the long 100-200mm of adult body, shell length is or greater than highly 2 times, is about 2.5-3 times of width.The shell front end is than taper, the wide circle in rear end; The shell table is more coarse, shell skin (the Wang Zhenrui that brown or Chestnut are arranged, China's fauna. Mollusca. Bivalvia: mussel order. the 1st edition. Beijing. Beijing Science Press, 1997) mussel shellfish meat meat contains rich nutrient substances, is the important source of high protein, vitamins D; Bibliographical information (Liu Zhifeng etc.; China's marine drug; 2001; 20 (6): 9) mussel has atherosclerosis; reducing blood-fat, thus the formation that hematoblastic cohesion suppresses thrombus suppressed, improve the supply and the protection expeirmental myocardial ischemia of myocardium oxygen and nutritive substance; microcirculation improvement and effect such as hypotensive, and the activeconstituents of this kind mussel is not appeared in the newspapers.
Summary of the invention
The present invention from the Mytilus crassitesta Lischke that grows in marine site, Chinese Zhejiang extraction separation to a kind of new polysaccharide with immuno-potentiation and antitumor action, called after MF4, through analysis of physical and chemical property, its physico-chemical property is: white cotton-shaped compound, soluble in water and dimethyl sulfoxide (DMSO), relative molecular weight is 113,000, mainly be made up of Fucose, seminose, glucose and semi-lactosi, ratio was followed successively by 15: 15: 26: 37; Glycosidic link be configured as beta comfiguration; The type of glycosidic link is the 1-4 glycosidic link, 1-2 glycosidic link and five-carbon sugar 1-3 glycosidic link, and ratio was followed successively by 80: 9: 2; And the glycosidic link that is present in the tapping point place is 1-6 glycosidic link and deoxidation hexose 1-2 glycosidic link, and ratio is 7: 10; End group sugar is except hexose, and molecule also contains part deoxidation hexose.
The extraction and separation method of MF4 MF4 of the present invention is as follows:
(1) extracts: after fresh Mytilus crassitesta Lischke shellfish meat is cleaned, blended with stirrer, heating was extracted 4 hours in 3 times of volume boiling water, extracting solution is with the ethanol sedimentation of 2 times of volumes 95%, precipitation is washed respectively 3 times with ethanol, acetone, removes albumen 2 times with the Savager method and (will precipitate and Savage reagent: CHCl
3-n-BuOH=4: 1 abundant mixing, centrifugal collection supernatant liquor is removed the protein denaturation layer between water and the organic phase), collect water, lyophilize gets the MF4 crude extract.
(2) separate: above-mentioned polysaccharide crude extract separates with DEAE-Mierocrystalline cellulose-52 (Pharmacia company), uses 0.05,0.1,0.2,0.5 successively, the NaCl wash-out of 1.0mol/L, the wash-out part of collecting 0.5mol/L, desalination and concentration, lyophilize, the MF part; MF separates with dextrane gel Sephadex G-100 (Pharmacia company), with the NaCl wash-out of 0.05mol/L, collects first elution peak, desalination and concentration, lyophilize; Handle with HPLC at last, (contain 14.2g/L Na with the 0.1M phosphatebuffer buffer system
2SO
4And 0.5g/LNaN
3The NaH of 0.1mol/L
2PO
4-Na
2HPO
4Damping fluid) as moving phase, collect first elution peak, desalination and concentration, lyophilize obtains polysaccharide MF4.
The analytical procedure and the result of MF4 MF4 physico-chemical property of the present invention are as follows:
(1) evaluation of HPGFC method purity: analytical column is TSK-G3000SWXL, 7.8mm * 30cm (Merck company), and moving phase is for containing 14.2g/L Na
2SO
4And 0.5g/L NaN
3The NaH of 0.1mol/L
2PO
4-Na
2HPO
4Damping fluid, sample size 50 μ l, flow velocity is 0.8ml/min, differential detects.The result shows: polysaccharide MF4 is single symmetrical peak, shows this uniformity of sample.
(2) mensuration of HPGFC method relative molecular weight: condition is the same with the evaluation of HPGFC method purity, with Dextran T10, T40, T70, T100, T120 is standard molecular weight, record elution volume Ve separately, record its void volume Vo with glucose T2000 (Pharmacia company), glucose records the cumulative volume Vt of post, with (Ve-Vo)/(Vt-Vo) to the logarithm of its molecular weight map typical curve, record the MF4 elution volume with same condition and method again, obtaining its relative molecular weight is 113,000.
(3) UV spectrum: MF4 does not have protein and nucleic acid charateristic avsorption band through UV scanning between 250-280nm.
(4) infrared spectra: the MF4 infrared spectra demonstrates the charateristic avsorption band of polysaccharide, 3442cm
-1(OH stretching vibration), 1650cm
-1(amide group C=O stretching vibration), 1137cm
-1, 995cm
-1(this broad peak is the angle vibration of the absorption peak and the hydroxyl of glycosidic link).
(5)
1HNMR: the polysaccharide proton signal is heaped in the narrow range of δ 4.0-5.51 mostly, except that the signal of anomeric proton δ 4.8-5.5 more easily resolves, the signal of other H2-H6 all concentrates between the δ 4.0-4.8, resolves difficulty.Usually the δ value of α-pyranohexose anomeric proton often surpasses 5.0, and the δ value of β type is then less than 5.0.The H-1 proton signal of MF4 is not obvious, δ should be less than 5.0 and the signal of the proton of other H2-H6 and heavy water overlapped, so think β-pyranohexose.
(6) proximate analysis: the MF4 H of 1mol/L
2SO
4Hydrolysis is made sugared nitrile acetic ester derivative and is used for the GC-MS analysis after 8 hours, show that with standard control its composition and ratio are Fucose: seminose: glucose: semi-lactosi=15: 15: 26: 37.The qualitative reaction of sulfuric acid carbazole method uronic acid is negative.
(7) glycosidic link analytical results: periodate oxidation reaction back adding phenolphthalein indicator presents colourless, illustrates that no formic acid generates, thereby judges that it does not contain the 1-6 glycosidic link.Carry out sugared nitrile acetylize GC-MS after the Smith degraded and analyze, find to have only glycerine and tetrahydroxybutane to exist, do not have monose to exist, illustrate and contain 1-4 glycosidic link and 1-2 glycosidic link, and do not contain the 1-3 glycosidic link.The polysaccharide MF4 posthydrolysis that methylates is made sugar alcohol acetyl derivatives GC-MS and analyze to be found that it contains: 1, and 5-two-O-ethanoyl-6-takes off O-2,3,4-three-O-methyl hexitol, 1,5-two-O-ethanoyl-2,3,4,6-four-O-methyl hexitol, 1,3,5-three-O-ethanoyl-2,4-two-O-methylpent sugar alcohol, 1,2,4,5-four-O-ethanoyl-6-takes off O-3-O-methyl hexitol, 1,2,5-three-O-ethanoyl-3,4,6-three-O-methyl hexitol, 1,4,5-three-O-ethanoyl-2,3,6-three-O-methyl hexitol, 1,4,5,6-four-O-ethanoyl-2,3-two-O-methyl hexitol, 1,4,5-three-O-ethanoyl-2-acetamido-3,6-two-O-methyl hexitol, and ratio is followed successively by: 8: 7: 1: 8: 7: 59: 6: 4, confirmed periodate oxidation and Smith degradation analysis result, and illustrate that also the type of this polysaccharide glycosidic link is the 1-4 glycosidic link simultaneously, 1-2 glycosidic link and five-carbon sugar 1-3 glycosidic link, ratio was followed successively by 80: 9: 2; And the glycosidic link that is present in the tapping point place is 1-6 glycosidic link and deoxidation hexose 1-2 glycosidic link, and ratio is 7: 10; End group sugar is except hexose, and the contained part deoxidation hexose of intramolecularly also is in the position of end group sugar.
This polysaccharide for first from Mytilus crassitesta Lischke shellfish meat extraction separation to, called after MF4.
The present invention has carried out external and intravital immune-enhancing activity experiment to polysaccharide MF4, shows significantly to promote NK cell activity and lymphocytic propagation, can improve functions of immune system.Animal vivo test shows that S180 sarcoma and Lewis lung cancer are all had significant inhibitory effect.So can be used for preparing immunostimulant or antitumor drug.
Embodiment:
Below in conjunction with embodiment the present invention is explained in detail.
Embodiment 1. preparation MF4 MF4
Selection is grown in the new scallop meat of the Mytilus crassitesta Lischke 16kg in marine site, Zhejiang, after cleaning, blending with stirrer, heating was extracted 4 hours in 3 times of volume boiling water, extracting solution is with the ethanol sedimentation of 2 times of volumes 95%, precipitation is washed respectively 3 times with ethanol, acetone, remove albumen 2 times with the Savager method, lyophilize gets the MF4 crude extract.The polysaccharide crude extract separates with DEAE-Mierocrystalline cellulose-52, uses 0.05,0.1,0.2,0.5 successively, the NaCl wash-out of 1.0mol/L, the wash-out part of collecting 0.5mol/L, lyophilize, the MF part.MF separates with dextrane gel (Sephadex G-100), with the NaCl wash-out of 0.05mol/L, collects first elution peak, handle with HPLC at last, as moving phase, collect first elution peak with the 0.1M phosphate buffered saline buffer, separation and purification obtains polysaccharide MF4 sample 217mg.
Embodiment 2. MF4 MF4 inside and outside enhancing immunity activity tests
1.T, bone-marrow-derived lymphocyte proliferative response experiment
Add mitogen concanavalin A (ConA) 5mgL routinely
-1Inducer T lymphocyte propagation adds mitogen lipopolysaccharides (LPS) 10mgL
-1Induce bone-marrow-derived lymphocyte propagation, a series of variation can take place in the form of cell and metabolism, is converted into parent cell, and differentiation and proliferation.The synthetic increase of intracellular protein and nucleic acid, and discharge multiple lymphokine.With
3H TdR mixes the influence of standard measure working sample on cell proliferation: add in nutrient solution
3H TdR participates among the new synthetic DNA it, quantitative DNA synthetic intensity in the cell, thus measure the propagation of cell, it is accurate and objective that radionuclide participates in method.With the toxicity of tetramethyl-azo azoles salt (MTT) method test sample pair cell, the plastosome deoxygenase can be reduced into blue first the part between the ribs and the hips by yellow with MTT in the viable cell, and first the part between the ribs and the hips output is directly proportional with viable cell.Behind organic solvent dissolution first the part between the ribs and the hips, available microplate reader detects optical density(OD) (OD) value.
Result: see table 1 for details, table 2
Table 1 polysaccharide MF4 sample is to T lymphocytotoxicity experimental result
Sample name concentration (mgL
-1) the OD value (cytotoxicity of x ± s)
Negative control 5 0.395 ± 0.001-
Polysaccharide MF4 1 0.502 ± 0.008 does not have
10 0.606 ± 0.012 does not have
100 0.814 ± 0.013 does not have
Annotate: if the OD value of experimental group is greater than the OD value of reference substance, with regard to the judgement sample nontoxicity
Table 2 polysaccharide MF4 is to T, bone-marrow-derived lymphocyte proliferative response result
T lymphocyte bone-marrow-derived lymphocyte
The thin B lymph of T lymph is thin
Concentration C onA irritating concentration LPS stimulates
The sample name
Born of the same parents comprehensively live
Born of the same parents comprehensively live
(mgL
-1) average (mgL of CPM
-1) CPM is average
Property evaluation (%)
Property evaluation (%)
Value (x ± s) be worth (x ± s)
Negative control 1966 ± 341 2013 ± 184
Positive control 5 29450 ± 1,429 10 24409 ± 1367
Polysaccharide MF4 1 41715 ± 2,020 42 1 33820 ± 1,659 39
10 41135±526 40 10 43241±1245 77
100 40935±2766 39 100 50974±3083 109
Annotate: test system: external
(1) result evaluation: lymphocytic propagation employing sample pulse index (CPM) value deducts the CPM value in positive control hole, and is on duty with % divided by the CPM in positive control hole.
(2) there is the negative sign representative sample that T, bone-marrow-derived lymphocyte are had restraining effect before the per-cent
(3) there is not the negative sign representative sample that T, bone-marrow-derived lymphocyte are had enhancement before the per-cent
(4) being judged as of lymphocytotoxicity " having " or " nothing "
(5) under the situation of acellular poison, the per-cent of the enhancing/inhibition of T, bone-marrow-derived lymphocyte is more than ± 15%, and expression has effect
(6) T lymphocyte positive control is concanavalin A (ConA), and the bone-marrow-derived lymphocyte positive control is lipopolysaccharides (LPS)
Above-mentioned experiment shows that MF4 all has tangible proliferation function to T, bone-marrow-derived lymphocyte.
2. MF4 MF4 is to the experiment of conversion of lotus Lewis lung cancer knurl mouse lymphocyte and NK cytoactive
Given the test agent: accurately take by weighing given the test agent MF4 and dilute with physiological saline, be mixed with each grade solution of desired concn, every mouse volume injected every day is 0.5ml.
Positive reference substance: Cyclophosphamide for injection (CTX), Hua Lian, Shanghai pharmaceutical factory produces.Polysaccharide-peptide capsule, Shanghai Xinkang Pharmaceutical Factory produces.
The knurl source: Mice Bearing Lewis Lung Cancer cell and mouse L929 culturing cell cultivate by pharmacological room of Shanghai Institute of Pharmaceutical Industry and interior generation is kept.
Laboratory animal: C
57The BL/6 mouse is provided by Shanghai Chinese Academy of Sciences Experimental Animal Center, conformity certification number: SCXK (Shanghai) 2003-0003.Body weight: 19-21g.Sex: male.Number of animals: every group of 6 mouse of experimental group and positive controls, negative control is 10.
Dosage is provided with: 5mg/kg/d.
Dosage regimen: intravenously administrable, every day 1 time, continuous 10 days.
Experiment contrast: negative control is given and the isopyknic physiological saline of experimental group, and dosage regimen is an intravenously administrable, every day 1 time, continuous 10 days.Positive control endoxan (CTX) 30mg/kg, intraperitoneal administration, every day 1 time, continuous 7 days.With reference to sample krestin 2g/kg, gastric infusion, every day 1 time, continuous 10 days.
(1) to lotus Lewis lung cancer C
57The lymphocyte proliferation assay of BL/6 mouse: get C
57The aseptic Lewis lung cancer suspension 0.05ml/ mouse (1 * 10 of making of BL/6 mouse toes subcutaneous vaccination
6Individual tumour cell).Next day, random packet was pressed the administration of experimental design scheme, and after the last administration, next day, mouse was respectively organized in dissection, and the aseptic spleen of winning is prepared into single splenocyte suspension with 100 eye mesh screens, and regulating cell concn with the RPM1640 nutrient solution that contains 10% deactivation calf serum is 1 * 10
7Individual cell/ml adds 96 porocyte culture plates with cell suspension, every hole 100 μ l (1 * 10
6Individual cells/well), adds the nutrient solution 100 μ l that contain ConA (5 μ g/ml) again, put 37 ℃, 5%CO
2Condition was cultivated after 72 hours, and gentle aspiration supernatant liquor 100 μ l add MTT staining fluid (5mg/ml, 20 μ l), put 37 ℃, 5%CO
2Condition is cultivated after 2 hours again and to be added Digestive system 100 μ l, measures the OD value in each hole next day, by following formula calculating stimulation index: stimulation index=experimental group OD value/control group OD value.
(2) to the test of the NK cytoactive of lotus Lewis lung cancer mouse: get C
57The aseptic Lewis lung cancer suspension 0.05ml/ mouse (1 * 10 of making of BL/6 mouse toes subcutaneous vaccination
6Individual tumour cell).Next day random packet, by the administration of experimental design scheme, after the last administration, next day, mouse was respectively organized in dissection, the aseptic spleen of winning is prepared into single splenocyte suspension with 100 eye mesh screens, the hypotonic red corpuscle of removing changes cell suspension in the culturing bottle over to 37 ℃ 5% CO
2Cultivate and remove attached cell after 1 hour, living cell counting also transfers to 3 * 10 with nutrient solution
6Individual cell/ml, the action effect cell.Target cell goes the conventional cultivation of L929 cultured cell in vitro 24 hours, and adjusting cell concn with nutrient solution is 1.5 * 10
5Individual cell/ml, making the ratio of imitating target cell is 20: 1.Get 96 well culture plates and add effector cell and target cell respectively, laying effect cell and target cell contrast in addition, in 37 ℃, 5%CO
2Condition was cultivated after 4 hours, add the MTT staining fluid, cultivate after 2 hours again and to add Digestive system and measure the OD value in each hole next day, by following formula calculating NK cell activity: NK cell toxicant %=[target cell contrasts OD average-(experimental group OD average-effector cell contrasts the OD average)]/target cell control group OD average * 100%
Experimental result sees table 3 and table 4 for details:
Table 3 polysaccharide MF4 influences lotus Lewis lung cancer mouse (subcutaneous vaccination) NK cytoactive
Group dosage regimen number of animals the weight of animals OD value
*The NK activity
(mg/kg/d) beginning/whole X ± SD at (only) beginning/end (g) (%)
MF4 5 iv×7qd 6/6 20.3/23.9 0.397±0.14
*** 55.57
CTX 30 ip×7qd 6/6 20.4/24.4 0.505±0.08 40.94
Polysaccharide-peptide 2000 ig * 10qd 6/6 20.2/20.5 0.358 ± 0.05
* *58.13
Negative control coordinative solvent iv * 7qd 10/10 20.4/24.7 0.517 ± 0.04 39.53
Annotate: experimental group is compared with negative control group,
* *Expression P value＜0.01
*Be experimental group OD average-effect control group OD average
The positive contrast of polysaccharide-peptide, CTX is the tumor chemotherapeutic drug endoxan
Table 4 MF4 is to lotus Lewis lung cancer C
57BL/6 mouse (subcutaneous vaccination) lymphocyte transformation increment influence
Group dosage regimen number of animals the weight of animals OD value
*Stimulation index
(mg/kg/d) (only) beginning/end (g) beginning/whole X ± SD
MF4 5 iv×7qd 6/6 20.3/23.9 0.947±0.07
*** 1.77
CTX 30 ip×7qd 6/6 20.4/24.4 0.463±0.05 0.87
Polysaccharide-peptide 2000 ig * 10qd 6/6 20.2/20.5 0.947 ± 0.07
* *1.79
Negative control coordinative solvent iv * 7qd 10/10 20.4/24.7 0.535 ± 0.06
Annotate: experimental group is compared with negative control group,
* *Expression P value＜0.01
Above-mentioned experiment shows, MF4 all is greatly increased and promoter action to lotus Lewis lung cancer NK cells in mice activity and lymphocyte transformation, have same value-added effect with the antitumor drug polysaccharide-peptide of clinical use, chemotherapeutics CTX then has the obvious suppression effect to lymphocytic conversion.
3. MF4 MF4 anti-tumor in vivo experiment
Experiment is prepared and design
Given the test agent: accurately take by weighing given the test agent MF4 and dilute with physiological saline, be mixed with each grade solution of desired concn, every mouse volume injected every day is 0.5ml.
Positive reference substance: lentinan for injection, Nanjing Zhenzhong Biological Engineering Co., Ltd produces.
Knurl source: murine sarcoma S-180 culturing cell, Mice Bearing Lewis Lung Cancer in-vivo tumour model; Keep by cultivation of pharmacological room of Shanghai Institute of Pharmaceutical Industry and interior generation.
Laboratory animal: C
57The BL/6 mouse is provided by Shanghai Chinese Academy of Sciences Experimental Animal Center, conformity certification number: SCXK (Shanghai) 2003-0003 number.Kunming mice is provided by Shanghai Institute of Pharmaceutical Industry's animal groups, conformity certification number: the card word is closed No. 117 in Shanghai.Body weight: 19-21g.Sex: female.Number of animals: every group of 10 mouse of experimental group and positive controls, negative control respectively is 20.
Dosage is provided with: high, medium and low dosage group is respectively 12.5mg/kg, 5mg/kg, 2mg/kg
Dosage regimen: intravenously administrable, every day 1 time, continuous 15 days.
Experiment contrast: negative control is given and the isopyknic physiological saline of experimental group, and dosage regimen is an intravenously administrable, every day 1 time, continuous 15 days.Positive control lentinan 2mg/kg, intravenously administrable, every day 1 time, continuous 15 days.
Experimental procedure
The murine sarcoma S-180 cell of taking the logarithm under the aseptic condition and growing dilutes into about 7 * 10 with physiological saline
6Individual/homogenate of ml cancer cells, oxter subcutaneous vaccination (0.2ml/ mouse) respectively, inoculate after 24 hours by the administration of experimental design scheme, get when experiment finishes and respectively organize tumour and negative control group contrast, be calculated as follows the tumour inhibiting rate of each group: tumour inhibiting rate %=[(negative control group gross tumor volume-administration group gross tumor volume)/the negative control group gross tumor volume] * 100%
Efficacy experiment to the inoculation of Lewis lung cancer toes: getting eugonic Lewis lung cancer knurl source under the aseptic condition, is solvent with physiological saline, adopts the preparation of homogenate method into about 1 * 10
7Individual/the ml cell suspension, the every mouse toes of corresponding host subcutaneous vaccination 0.05ml/ mouse, next day is by the administration of experimental design scheme, two weeks back execution animal, the intercepting toes are weighed, and are calculated as follows the tumour inhibiting rate of each group: the average lotus knurl of tumour inhibiting rate %=[(negative control group average lotus knurl toes weight-administration group toes are heavy)/(the average toes of the average lotus knurl of negative control group toes weight-blank group are heavy)] * 100%
Experimental result: MF4 is with high, medium and low dosage group 12.5mg/kg, 5mg/kg, and 2mg/kg dosage iv * 15qd scheme is respectively 61.19%, 55.24%, 39.16% to the tumour inhibiting rate of mouse S-180 sarcoma oxter subcutaneous vaccination, sees table 5 for details; Tumour inhibiting rate to the subcutaneous vaccination of Mice Bearing Lewis Lung Cancer toes is respectively 49.34%, 43.41%, 34.73%, sees Table 6.
Table 5 MF4 polysaccharide is to the curative effect of Kunming mouse sarcoma S-180 (armpit subcutaneous vaccination) model
The heavy tumour inhibiting rate of sample dose dosage regimen number of animals the weight of animals knurl
(mg/kg/d) (g) X ± SD at (g) beginning/end at (only) beginning/end (%)
MF4 12.5 iv×15qd 10/10 19.5/26.3 1.11±0.17
*** 61.19
MF4 5 iv×15qd 10/10 19.8/26.9 1.28±0.19
*** 55.24
MF4 2 iv×15qd 10/10 19.3/27.1 1.74±0.17
*** 39.16
Lentinan 2 iv * 15qd 10/10 19.6/26.5 1.92 ± 0.13
* *32.87
Negative control coordinative solvent iv * 15qd 20/20 19.4/27.9 2.86 ± 0.26
Annotate: experimental group is compared with negative control group, and * * * represents P value＜0.01
Table 6 MF4 polysaccharide is to the curative effect of Kunming mouse Lewis lung cancer (toes subcutaneous vaccination) model
Heavy (g) tumour inhibiting rate of sample dose dosage regimen number of animals the weight of animals knurl
(mg/kg/d) beginning/whole X ± SD at (only) beginning/end (g) (%)
MF4 12.5 iv×15qd 10/10 20.9/25.6 0.461±0.09
*** 49.34
MF4 5 iv×15qd 10/10 20.4/26.1 0.515±0.09
*** 43.41
MF4 2 iv×15qd 10/10 20.5/25.9 0.594±0.12
*** 34.73
Lentinan 2 iv * 15qd 10/10 20.6/26.3 0.680 ± 0.13 25.27
CTX 30 ip×7qd 10/10 20.6/24.2 0.024±0.02
*** 97.36
Negative control coordinative solvent iv * 15qd 20/20 20.6/26.5 0.91 ± 0.14
Annotate: experimental group is compared with negative control group,
* *Expression P value＜0.01
The result shows that the MF4 intravenously administrable all has obvious antitumor curative effect to Mice Bearing Lewis Lung Cancer and S-180 sarcoma, and shows and certain dose-effect relationship also be proportionate with the course of treatment.The weight of animals all has more significantly growth before and after each experimental group experimentation on animals, points out MF4 toxicity of the present invention less.With the lentinan of present clinical use relatively, under Isodose, MF4 to the inhibiting rate of tumour apparently higher than lentinan.Therefore, MF4 MF4 can be used for preparing new immunostimulant preparation or antitumor drug.The present invention has opened up new approach to the Living marine resources that develop China.
Claims (3)
1. a MF4 MF4 is characterized by white floss, soluble in water and dimethyl sulfoxide (DMSO), and relative molecular weight is 113,000, mainly is made up of Fucose, seminose, glucose and semi-lactosi, ratio was followed successively by 15: 15: 26: 37; Glycosidic link be configured as beta comfiguration; The type of glycosidic link is the 1-4 glycosidic link, 1-2 glycosidic link and five-carbon sugar 1-3 glycosidic link, and ratio was followed successively by 80: 9: 2; And the glycosidic link that is present in the tapping point place is 1-6 glycosidic link and deoxidation hexose 1-2 glycosidic link, and ratio is 7: 10; The end group steamed bun stuffed with sugar contains hexose and part deoxidation hexose.
2. the preparation method of the described polysaccharide MF4 of claim 1, its concrete steps are as follows:
(1) extracts: after fresh Mytilus crassitesta Lischke shellfish meat is clean, stirrer blends, heating was extracted 4 hours in 3 times of volume boiling water, extracting solution is with the ethanol sedimentation of 2 times of volumes 95%, precipitation is washed respectively 2 times with ethanol, acetone, the Savager method is removed albumen 2 times, collect water, lyophilize gets the MF4 crude extract;
(2) separate: above-mentioned polysaccharide crude extract by DEAE-Mierocrystalline cellulose-52 post, uses 0.05,0.1,0.2,0.5 with water dissolution successively, the NaCl wash-out of 1.0mol/L, the wash-out part of collecting 0.5mol/L, desalination and concentration, lyophilize, the MF part; MF with the NaCl wash-out of 0.05mol/L, collects first elution peak, desalination and concentration, lyophilize by Sephadex G-100 sephadex column; Use the HPLC purifying at last, (contain 14.2g/L Na with the 0.1M phosphatebuffer buffer system
2SO
4And 0.5g/L NaN
3The NaH of 0.1mol/L
2PO
4-Na
2HPO
4Damping fluid) as moving phase, collect first elution peak, desalination and concentration, lyophilize obtains polysaccharide MF4.
3. the application of the described polysaccharide MF4 of claim 1 in preparation immunostimulant or antitumor drug.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007041950A1 (en) * | 2005-10-11 | 2007-04-19 | Dalian Polytechnic University | Extraction method of patinopecten yessoensis polysaccharide |
| CN100354310C (en) * | 2005-10-11 | 2007-12-12 | 大连轻工业学院 | Mussel polysacharide and its preparing method |
| CN100486593C (en) * | 2005-03-15 | 2009-05-13 | 中国人民解放军第二军医大学 | Trachyostracous mussel extract capable of increasing immunity function |
| CN101357952B (en) * | 2008-09-18 | 2011-03-23 | 中国人民解放军第二军医大学 | Polysaccharide MA from Mytilus coruscus with hypolipidemic activity and preparation method thereof |
| CN103204950A (en) * | 2013-04-23 | 2013-07-17 | 李苹苹 | Method for purifying polysaccharide compound in mussel cooking juice |
| CN107227327A (en) * | 2017-07-07 | 2017-10-03 | 山东省药学科学院 | A kind of preparation method of mussel oligosaccharides and products thereof and application |
| CN109553696A (en) * | 2018-12-22 | 2019-04-02 | 大连海洋大学 | A kind of preparation method of mussel class cooking liquor polysaccharide |
| CN111544587A (en) * | 2020-06-29 | 2020-08-18 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and application thereof |
| WO2022000205A1 (en) * | 2020-06-29 | 2022-01-06 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and use thereof |
-
2004
- 2004-06-07 CN CN 200410024958 patent/CN1273496C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100486593C (en) * | 2005-03-15 | 2009-05-13 | 中国人民解放军第二军医大学 | Trachyostracous mussel extract capable of increasing immunity function |
| WO2007041950A1 (en) * | 2005-10-11 | 2007-04-19 | Dalian Polytechnic University | Extraction method of patinopecten yessoensis polysaccharide |
| CN100354310C (en) * | 2005-10-11 | 2007-12-12 | 大连轻工业学院 | Mussel polysacharide and its preparing method |
| CN101357952B (en) * | 2008-09-18 | 2011-03-23 | 中国人民解放军第二军医大学 | Polysaccharide MA from Mytilus coruscus with hypolipidemic activity and preparation method thereof |
| CN103204950A (en) * | 2013-04-23 | 2013-07-17 | 李苹苹 | Method for purifying polysaccharide compound in mussel cooking juice |
| CN107227327A (en) * | 2017-07-07 | 2017-10-03 | 山东省药学科学院 | A kind of preparation method of mussel oligosaccharides and products thereof and application |
| CN107227327B (en) * | 2017-07-07 | 2020-08-18 | 山东省药学科学院 | Preparation method of mussel oligosaccharide, and product and application thereof |
| CN109553696A (en) * | 2018-12-22 | 2019-04-02 | 大连海洋大学 | A kind of preparation method of mussel class cooking liquor polysaccharide |
| CN111544587A (en) * | 2020-06-29 | 2020-08-18 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and application thereof |
| WO2022000205A1 (en) * | 2020-06-29 | 2022-01-06 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and use thereof |
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| Publication number | Publication date |
|---|---|
| CN1273496C (en) | 2006-09-06 |
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