CN102134284A - Anti-hepatoma drug strongylocentrotus nudus egg polysaccharide SEP-1 based on immune regulation - Google Patents
Anti-hepatoma drug strongylocentrotus nudus egg polysaccharide SEP-1 based on immune regulation Download PDFInfo
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Abstract
The invention relates to a strongylocentrotus nudus egg polysaccharide SEP-1 effectively resisting mouse hepatoma cells, which has the molecular weight of 1.65*106Da. The SEP-1 sequentially has the inhibition ratio of 43.2%, 52.8% and 60.1% on transplantable tumors of mouse hepatoma cells H22 under the dosages of 4 mg/kg/d, 8 mg/kg/d and 16 mg/kg/d and does not have obvious influence on the weight of H22 tumor-bearing mice so as to indicate that the strongylocentrotus nudus egg polysaccharide SEP-1 with uniform components has the advantages of outstanding mouse hepatoma resistant effect, low toxic and side effects, little adverse reaction, and the like; and the hepatoma resistant effect of the strongylocentrotus nudus egg polysaccharide SEP-1 is based on activating the cellular immunity and the humoral immunity of organisms so as to show that the strongylocentrotus nudus egg polysaccharide SEP-1 has wide application prospect on the aspects of tumor treatment and organism immunocompetence regulation.
Description
Technical field:
The present invention relates to a kind of based on immunoregulatory medicines resistant to liver cancer gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1.
Background technology:
Sea urchin is a kind of echinoderms, mainly contains kinds such as Anthocidaris crassispina, shrimp pancreas horsedung sea urchin and sour jujube hat sea urchin.Sea urchin contains nutritive ingredients such as rich in protein, polysaccharide, lipid acid, VITAMIN and trace element, and its edible portion is its endoceliac sexual gland (ovary, a spermary), and the general designation sea urchin egg claims gonad of Hemicentrotus seu Strongylocentrotus or sea urchin cream again; The shell of sea urchin and quil are important simply Chinese medicine, has multiple medicinal efficacy, " book on Chinese herbal medicine is original " calls it the effect of " controlling distressed ", the function that " Chinese medicinal herbal " master is carried it to be had " softening and resolving hard mass, cards such as the detumescence of reducing phlegm, the scrofula that disappears subcutaneous nodule, long-pending phlegm are not changed, distending pain in the chest and hypochondrium ".This experiment to the ovum (gonad of Hemicentrotus seu Strongylocentrotus) of Strongylocentrotus nudus Strongylocentrotus nudus when studying extraction separation arrived the gonad of Hemicentrotus seu Strongylocentrotus polysaccharide, studies show that in recent years and contain antitumor and immunologic active material in the sea urchin.
The class natural macromolecular compound that aldose that polysaccharide is coupled together by glycosidic link or ketose are formed, scientists is found from a large amount of pharmacology and clinical study, a kind of often immunomodulator of the polysaccharide of from natural product, separating, its can immune cell activated and normal cell is not had toxic side effect; In treatment during tumour, it directly kills the cancer cells of growth unlike chemotherapeutics, but as a kind of biological immune reaction or add that powerful antibody generates and activator complement etc., reaches the effect that suppresses and eliminate tumour cell, and is minimum to Normocellular influence.Along with deepening continuously of polysaccharide research, lentinan entered clinical application in 1978, and as the ancillary drug of immunomodulator and tumor chemoradiotherapy treatment, effect is remarkable.At present, a lot of bioactive polysaccharides are as the immunoregulatory activity and the anti-tumor activity of the polysaccharide of separation and Extraction from multiple fungies such as rainbow conk, Poria cocos, umbellate pore furgus, glossy ganoderma and many higher plants, the equal tool wide spectrum of animal polysaccharide.Studies show that in a large number polysaccharide also has decreasing cholesterol, hypoglycemic, anti-oxidant, anti-inflammatory, antiviral isoreactivity.
The gonad of Hemicentrotus seu Strongylocentrotus polysaccharide system separation and purification from the Strongylocentrotus nudus Huang that the present invention relates to obtains homogeneous polysaccharide component S EP-1, and the high performance liquid phase result shows that SEP-1 is a molecular weight 1.65 * 10
6The polysaccharide of Da.Pharmacodynamic study is the result show, gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 has remarkable restraining effect to rat liver cancer cell H22 transplanted tumor.Gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 of the present invention does not report as yet based on the pharmacological action of immunoregulatory anti-liver cancer.
Summary of the invention:
The application of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide of the present invention in preparation antitumor drug and adjusting body's immunity medicine.
The invention provides the preparation method of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide and determining of relative molecular weight; Pharmacodynamic experiment shows, the gonad of Hemicentrotus seu Strongylocentrotus polysaccharide has anti-rat liver cancer growth effect in the body, and external the H22 cell do not had tangible cytotoxicity.
Description of drawings:
The S-400 molecular sieve elution curve of Fig. 1 gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides.
The HPLC of Fig. 2 gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1.
Fig. 3 gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to the body weight provide protection of H22 mice with tumor.
The collaborative ConA/LPS of Fig. 4 gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 stimulates the spleen lymphocyte proliferation effect.
Embodiment:
The objective of the invention is to study a kind of molecular weight that extracts from gonad of Hemicentrotus seu Strongylocentrotus and be separated to is the homogeneous polysaccharide of XX, and its anti-tumor activity is not reported as yet, below preparation and the resisting liver cancer activity of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 done elaboration:
Embodiment 1: the preparation method of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1
A. get fresh gonad of Hemicentrotus seu Strongylocentrotus and add anhydrous propanone processing 6 times, each half an hour,, obtain the exsiccant gonad of Hemicentrotus seu Strongylocentrotus the acetone evaporate to dryness;
B. cross with 90 ℃ of hot water lixiviate acetone treatment and exsiccant gonad of Hemicentrotus seu Strongylocentrotus 6 hours, totally 3 times, merge vat liquor, vat liquor is concentrated, filter, filtrate;
C. adjust gonad of Hemicentrotus seu Strongylocentrotus extracting solution pH6.5, add 0.5% papoid, 60 ℃ of incubated overnight, 90 ℃ of heating 15min make enzyme deactivation, centrifugal collection supernatant liquor;
D. till the extracting solution behind the enzymolysis does not have a precipitation with Sevag reagent Deproteinization to the interface between chloroform and the water repeatedly;
E. the polysaccharide extraction liquid behind the Deproteinization is evaporated to proper volume, slowly adds the dehydrated alcohol of 4 times of volumes, 4 ℃ of placements are spent the night, centrifugal collecting precipitation, and vacuum-drying obtains gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides (EWP).
F. with dried raw sugar, water-soluble and ultrafiltration obtains the bigger gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides (EWP-H) of molecular weight, it is made into the aqueous solution of 20mg/mL, last DE52 cellulose ion exchange column is with the NaCl solution gradient wash-out of 0～1.0mol/L, flow velocity 1.0mL/min, every pipe 5mL fraction collection, the phenolsulfuric acid method is measured sugared content by pipe, draws elution curve, merges same composition;
G. the component that ion-exchange is collected is through Sephacryl 400 posts, with the NaCl eluant solution of 0.05mol/L, and flow velocity 0.45mL/min, detection method is the same, draws elution curve and obtains two peaks, intercepts wherein molecular weight less than 2 * 10
6The component of Da, dialysis is concentrated, dry, gets gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 (accompanying drawing 1).
Embodiment 2: the mensuration of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 molecular weight
Waters 600 highly effective liquid phase chromatographic systems adopt Shodex KS-805 chromatographic column, 2414 differential refraction detectors, and moving phase is water, sample concentration is 5.0mg/ml, sample size 20ul, flow velocity 1.0ml/min, 30 ℃ of column temperatures, record sample chromatogram curve.
The result shows (accompanying drawing 2), and the appearance time of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is 6.102min, and purity is 90%, and to extrapolate its averagemolecular wt amount according to typical curve be 1.65 * 10
6Da.
SEP-1 produces tumor-inhibiting action by immunomodulatory in the body for checking gonad of Hemicentrotus seu Strongylocentrotus polysaccharide, has carried out six antitumor tests.
Embodiment 3: gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to the tumor-inhibiting action of H22 tumor-bearing mice
Set up the H22 bearing mouse model by the transplanted tumor organon.The mouse behind the H227d is inoculated in selection, the sterilization skin of abdomen, and with aseptic empty needle suction ascites, physiological saline dilution, trypan blue is counted under inverted microscope with blood counting chamber after dyeing, and viable count is more than 98%, and adjusting cell concn is 4 * 10
7/ ml.60 of 18-20g ICR mouse are adopted in experiment, are divided into 6 groups at random: blank group, tumor model group, the basic, normal, high dosage treatment group of SEP-1, positive controls (endoxan: CTX), 10 every group, weigh and record.Except that the normal control group, respectively organize mouse right fore armpit subcutaneous vaccination 0.2mlH22 cell suspension in other.Begin administration behind the inoculation 24h, all to the physiological saline of equivalent, endoxan group dosage is 20mg/kg for normal control group and model group, and SEP-1 is basic, normal, high, and three dosage groups are respectively 4,8, and 16mg/kg/d, administering mode are tail vein injection (iv).Set time every day is administered once and writes down the active situation of mouse, 12d continuously.Weigh behind the last administration 24h, mouse is put to death in the cervical vertebra dislocation, gets knurl piece, spleen and thymus gland and weighs respectively, calculates tumour inhibiting rate, spleen index and thymus index.Formula is as follows:
The body weight (g) of heavy (the mg)/mouse of the spleen of spleen index=mouse
The body weight (g) of heavy (the mg)/mouse of the thymus gland of thymus index=mouse
Data represent that with X ± SD the t check the results are shown in Table 1 between group
Table 1SEP-1 to the influence of mouse bearing liver cancer cell H22 (n=10,
)
*P＜0.05,
*P＜0.01,
* *P＜0.001vs model group;
#P＜0.05,
##P＜0.01,
###P＜0.001vs positive controls
The result shows, basic, normal, high three dosage of SEP-1 are respectively 43.2%, 52.8%, 60.1% to the inhibiting rate of mouse transplanted sarcoma solid tumor H22, the tumor control rate of endoxan group is 78.0%, the SEP-1 high dosage can reach 77% of endoxan tumor killing effect, and effect is apparent in view.Compare with the endoxan group, SEP-1 does not make significant difference to the body weight of mouse, and can improve spleen index/thymus index to some extent, shows that immunomodulatory is the antineoplastic a kind of vital role mode of SEP-1.
Embodiment 4: gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to the influence of H22 tumor-bearing mice body weight
After the foundation of H22 tumor-bearing mice tumor model, during each administration group successive administration 12d, the body weight of mouse is respectively organized in weighing every day.With the administration time is X-coordinate, and the mouse body weight is total coordinate, draws the body weight change curve of H22 tumor-bearing mice.
The result shows (accompanying drawing 3), and blank group mouse body weight is normal ascendant trend, though positive chemotherapeutic tumor killing effect is remarkable, almost do not rise through the H22 of its treatment mice with tumor body weight, during the administration also with weight loss; And gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 during administration except having tumor killing effect preferably, also show body weight provide protection to the H22 mice with tumor, the tumor-inhibiting action that SEP-1 is described is that immunomodulatory realizes, body be there is no tangible injury.
Embodiment 5: the pathological examination of the H22 tumor-bearing mice tumor tissue of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 treatment
A same test of experimental animal and normal control group, tumor model group and drug treatment group.Experiment finishes, and puts to death mouse and takes out transplanted tumor, observes its tissue pathologies change.Tumor tissue is fixed through 10% formaldehyde solution, routine is drawn materials, dehydration, paraffin embedding, film-making (4 μ m are thick), HE dyeing, under opticmicroscope, read sheet by the pathology professional,, be labeled as "-", "+", " ++ ", " +++", " ++ ++ " expression successively according to pathology light and heavy degree difference, wherein "-" do not change for having obviously, " ++ ++ " be serious pathological change.
Tumor cell proliferation is active in the tumor tissue of model group mouse, visible more pathologic mitosis phase (+++), accidental little focal necrosis (+); And tumour cell volume-diminished (++) in the tumor tissue of low, the middle dosed administration group of SEP-1 mouse, tumour cell volume-diminished in the tumor tissue of high dosage administration group mouse (+++), pathologic mitosis is visible (+) mutually, and tumor tissues is seen focal necrosis (++).Tumour cell volume-diminished in the tumor tissue of positive chemotherapeutic endoxan group mouse (+++), pathologic mitosis is visible (+) mutually, the visible focal necrosis of tumor tissues (++).From The above results as can be seen, the degree of SEP-1 high dosage (16mg/kg/d) treatment group mouse tumor tissue necrosis is suitable with male treatment group effect, show comparatively significantly tumor-inhibiting action, to inhibitory rate 60.1% in the rat liver cancer cell H22 body, compare with other natural marine class or bioactive polysaccharide, show stronger tumor killing effect.
Embodiment 6: the collaborative ConA/LPS of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 stimulates the spleen lymphocyte proliferation experiment
A same test of experimental animal and normal control group, tumor model group and drug treatment group.Experiment finishes, the cervical vertebra dislocation is put to death and is respectively organized mouse, shred, grind and make the spleen single cell suspension in the aseptic taking-up spleen horizontalization ware, behind erythrocyte cracked liquid cracking 5min, centrifugal abandon supernatant after, after cleaning 2 times with physiological saline again, trypan blue dyeing counting (viable count＞95%) is adjusted cell concn to 5 * 10 with complete culture solution
6/ ml.In the 96 porocyte culture plates, ((the H22+1640 nutrient solution+ConA/LPS+SEP-1), each group is all established 3 multiple holes for H22+1640 nutrient solution+ConA/LPS), measuring hole to be provided with negative control hole (RPMI-1640), blank hole (H22+1640 nutrient solution), ConA or LPS control wells.Every hole adds splenic lymphocyte suspension 100 μ l (5 * 10
5Cells/well), then giving ConA (final concentration is 5 μ g/ml) or LPS (final concentration is 4 μ g/ml) stimulates, and culture plate is put 37 ℃, 5%CO
2Incubator in continue cultivation effect 24h after, add MTT, every hole 20 μ l, after continuing to cultivate 4h, the sucking-off nutrient solution adds DMSO150 μ l, surveys each hole light absorption value in the 570nm wavelength on the rearmounted microplate reader of concussion 10min.
The result shows (accompanying drawing 4), the OD value that basic, normal, high each the administration group of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is surveyed all is higher than the tumor model group, gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 (4,8 shown in Figure 3,16mg/kg/d) splenic lymphocyte to the H22 tumor-bearing mice has tangible proliferation function, and is dose-dependently; Simultaneously, and gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 (4,8,16mg/kg/d) can work in coordination with the ConA inducer T lymphocyte and significantly breed, and be dose-dependently; Gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 (4,8,16mg/kg/d) also can work in coordination with LPS and induce bone-marrow-derived lymphocyte significantly to breed, and be dose-dependently.
Prove that from the humoral immunization of cell-mediated cellular immunization of T and B mediation SEP-1 is at the intravital immunoregulation effect of H22 mouse respectively below.
Example 7: gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to the influence of H22 mice spleen supernatant T cell quantity
The spleen of aseptic each treated animal of taking-up is made a same test of single cell suspension, and concentration is adjusted into 5 * 10
6/ ml.Anti-mouse CD3, CD4, the CD8 monoclonal antibody of splenocyte and FITC mark are hatched 30min jointly in 30 ℃ of water-baths.Hatch finish after, with FAC damping fluid mixing gently, 3000r/min, centrifugal 5min abandons supernatant, behind the flushing cell 2 times, with FAC damping fluid re-suspended cell, cell suspension is moved in the FACS dedicated pipe, prepares to carry out instrument detecting and analysis.Detect the percentage composition (table 2) of the T cell subunit under the 525nm with flow cytometer.
The result shows that the basic, normal, high dosage group of SEP-1 all can raise CD4 in the H22 mice spleen supernatant
+T, CD8
+The percentage composition of T cell, and be dose-dependently.In the mouse boosting cell of this explanation SEP-1 treatment, the immunne response that the T cell of mediation specific immunity participates in plays an important role in killing tumor cell, control tumor growth, CD4 and the quantitative up-regulated of CD8 subgroup prove that SEP-1 can come immunity of organism is raised by the quantity of regulating the T cell.CD4
+The effect of T cell killing tumour cell mainly mediates by discharging cytokine activated mononuclear scavenger cell and NK cell, in some cases, and CD4
+The T cell also can directly dissolve target cell; CD8
+The T cell can be divided into CTL and directly bring into play killing tumor cell.
Table 2SEP-1 is to the influence of T cell subunit content in the H22 mouse boosting cell supernatant
*P＜0.05,
*P＜0.01,
* *P＜0.001vs model group;
#P＜0.05,
##P＜0.01,
###P＜0.001vs positive controls
Example 8: gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to the influence of Ig level in the H22 mice serum
Choose and above-mentionedly respectively organize laboratory animal and pluck the eyeball periphery and get blood, be collected into 1ml, centrifugal; Get serum 40 μ L respectively, 10 μ L, 1mlIgA respectively is equipped with in 80 μ L adding, and IgG is in the test tube of IgM, react 15min in the rearmounted 37 ℃ of water-baths of mixing, record the light absorption value of each sample under 340nm with the semiautomatic biochemistry detector,, read IgA with the physiological saline zeroing, IgG, the concentration value of IgM (g/L).
The result shows (table 3), and gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 is to IgA in the H22 mice serum, IgG, and the IgM expression level all has the rise effect, and is dose-dependently; And IgA in the H22 mice serum after the SEP-1 administration, IgG, the IgM expression level all is higher than normal mouse.Studies show that at present the more IgG that distributes in the body may be by following machine-processed killing tumor cells: the cytotoxicity that panimmunity cell performance antibody-dependant cell is mediated makes oncolysis; 2. by activating complement system dissolves tumour cell; 3. the IgG antibody-like also can play opsonization by the IgG Fc acceptor on phagocytic cell surface with after tumour cell combines, and promotes engulfing tumour cell; 4. at the antibody of tumor cell surface acceptor such as TfR, Transferrins,iron complexes capable of blocking promotes the effect of growth of tumour cell, thereby suppresses the growth of tumour cell.
Table 3SEP-1 is to the influence of Ig expression level in the H22 mice serum
*P＜0.05,
*P＜0.01,
* *P＜0.001vs model group;
#P＜0.05,
##P＜0.01,
###P＜0.001vs positive controls
Claims (4)
1. one kind has gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 of anti-tumor activity and immunoregulation effect and preparation method thereof, it is characterized in that may further comprise the steps:
A. get fresh gonad of Hemicentrotus seu Strongylocentrotus and add anhydrous propanone and handle, carry, obtain gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides (EWP) behind the Deproteinization, alcohol precipitation, vacuum-drying through water;
B. water-soluble and ultrafiltration obtains the bigger gonad of Hemicentrotus seu Strongylocentrotus Crude polysaccharides (EWP-H) of molecular weight with EWP, and behind DE52 cellulose ion exchange column, Sephacryl 400 posts, molecular weight is less than 2 * 10 in the intercepting elutriant successively
6The component of Da, dialysis is concentrated, dry, gets gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1.
2. the preparation method of gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 as claimed in claim 1 is characterized in that this gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 molecular weight for preparing is 1.65 * 10
6Da.
3. a gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 according to claim 2 has tumor killing effect and immunoregulation effect preferably to rat liver cancer cell H22.
4. resisting liver cancer activity and the mechanism research of the gonad of Hemicentrotus seu Strongylocentrotus polysaccharide SEP-1 based on immunoregulation effect according to claim 3.
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Cited By (3)
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| CN102680272A (en) * | 2012-05-29 | 2012-09-19 | 烟台渤海制药集团有限公司 | Extraction, purification and identification method for sea urchin polysaccharides |
| CN103382229A (en) * | 2013-06-21 | 2013-11-06 | 中国药科大学 | Preparation method and structural identification for novel strongylocentrotus nudus egg polysaccharide having immunoregulation effect |
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
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| CN101974097A (en) * | 2010-10-29 | 2011-02-16 | 中国药科大学 | Scale separation and purification method of pentaxanthin amylose |
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| CN101974097A (en) * | 2010-10-29 | 2011-02-16 | 中国药科大学 | Scale separation and purification method of pentaxanthin amylose |
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| 《中国海洋药物杂志》 20060630 刘纯慧,奚涛,林亲雄,高翼,邢莹莹 海胆黄多糖的分离、纯化及免疫活性测定 第2页左栏倒数第1段，第4页左栏"3.2 分离纯化"一节 1-2 第25卷, 第3期 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680272A (en) * | 2012-05-29 | 2012-09-19 | 烟台渤海制药集团有限公司 | Extraction, purification and identification method for sea urchin polysaccharides |
| CN103382229A (en) * | 2013-06-21 | 2013-11-06 | 中国药科大学 | Preparation method and structural identification for novel strongylocentrotus nudus egg polysaccharide having immunoregulation effect |
| CN103382229B (en) * | 2013-06-21 | 2016-01-13 | 中国药科大学 | A kind of preparation method and Structural Identification with the novel SEP-1 of immunoregulation effect |
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
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Application publication date: 20110727 |