CN105147720A - Application of crocodile blood polysaccharide to liver cancer treatment - Google Patents
Application of crocodile blood polysaccharide to liver cancer treatment Download PDFInfo
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Abstract
The invention provides application of crocodile blood polysaccharide to liver cancer treatment. The crocodile blood polysaccharide is a polysaccharide component purified from crocodile blood powder for the first time, and meanwhile crocodile blood polysaccharide has the close relationship with immune function regulation, cell and cell identification, intercellular substance transportation, cancer diagnosis and treatment, and the like, and has the antiviral, anti-tumor and immune regulation functions. Meanwhile, the crocodile blood polysaccharide component can be combined with acid, alkali, salt, aquo-complex or ester and other auxiliary materials on the aspect of pharmacy so as to be prepared into tablets, capsules, oral liquid preparations, emulsion, injections or combinations of tablets, capsules, oral liquid preparations, emulsion and injections, and when used as medicine for treating cancer or tumors, the crocodile blood polysaccharide has the obvious anti-tumor function, can effectively control growth of tumor cells, and is more beneficial to research on anti-tumor activity.
Description
Technical field
The invention belongs to medicinal chemistry art, be specifically related to the application of crocodile blood polysaccharide in liver cancer treatment.
Background technology
Crocodile is the most ancient reptile, has survived on earth more than 200,000,000 3 thousand ten thousand years, has thus had the title of " living fossil ".
Traditional Chinese medical science ancient book is recorded, and crocodile blood has the effects such as heat-clearing and toxic substances removing, antiinflammatory, treating tumor.In recent years, foreign scholar's effect to crocodile blood is studied, and existing crocodile blood has the report of the aspect effects such as antibacterial, antiviral, and contributes to promoting laboratory animal primary immune response ability under pathological conditions.Modern study shows, crocodile blood has two large significant functional characteristics: crocodile whole blood, hemoglobin, serum, blood plasma and leukocyte have antibacterium, fungus, virus and active anticancer.Another remarkable functional characteristics is, the oxygen carrying content of crocodile hemoglobin exceedes more than 100 times of other animal, this special hemoglobin can improve the blood oxygen carrying content of human body after entering human body and human body hemoglobin synthesis, enough oxygen is delivered to each position of brain and health, strengthen vigor and the mutation ability of body fluid and cell.
Why crocodile blood has so many effect, we think may have very large relation with the polysaccharide in blood, because large quantity research shows, the identification of the adjustment of polysaccharide and immunologic function, cell and cell, the transport of intercellular substance, the Clinics and Practices etc. of cancer have close relationship, have antiviral, antitumor and immunoregulation effect.Therefore we by the Polyose extraction in crocodile blood out, have carried out the research of anti-tumor activity.
Summary of the invention
In view of this, the present invention is intended to propose the application of crocodile blood polysaccharide in liver cancer treatment, to solve problems of the prior art.
For achieving the above object, technical scheme of the present invention is achieved in that
Crocodile blood polysaccharide is preparing the application in cancer treatment drug.
Further, the application in the medicine suppressing liver cancer growth or transfer is being prepared in described application.
Further, described crocodile blood polysaccharide is the mixture containing at least one polysaccharide component of gained after separation and purification from crocodile blood blood powder.
Further, described crocodile blood polysaccharide comprises 1 ~ 3 kind of polysaccharide component.
Further, the described pharmaceutical pack of Hepatoma therapy is containing crocodile blood polysaccharide and pharmaceutically acceptable acid, alkali, salt, hydrate or ester and other adjuvants.
Further, described medicine can be prepared at least one in tablet, capsule, oral liquid, emulsion and injection.
Relative to prior art, the crocodile blood polysaccharide application prepared in cancer treatment drug of the present invention has following advantage:
Described crocodile blood polysaccharide is the polysaccharide component of purifying from crocodile blood blood powder first, the identification of the simultaneously adjustment of described crocodile blood polysaccharide and immunologic function, cell and cell, the transport of intercellular substance, the Clinics and Practices etc. of cancer have close relationship, have antiviral, antitumor and immunoregulation effect; Meanwhile, described crocodile blood polysaccharide component pharmaceutically can with acid, alkali, salt, hydrate or ester; And the combination of other adjuvants is prepared into tablet, capsule, oral liquid, emulsion, injection or their combination, use as Therapeutic cancer or tumour medicine, have and significantly improve anti-tumor function, and effectively control the growth of tumor cell, advantageously in the research of anti-tumor activity.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 show the polysaccharide crude extract extracted in crocodile blood dry powder cross SephadexG-200 post after elution curve;
Fig. 2 shows MTT and to detect in crocodile blood polysaccharide three kinds of different components to the inhibitory action of BEL-7402 hepatoma cell proliferation;
Fig. 3 shows MTT and to detect in crocodile blood polysaccharide three kinds of different components to the inhibitory action of SMMC-7721 hepatoma cell proliferation;
Fig. 4 shows MTT and to detect in crocodile blood polysaccharide three kinds of different components to the inhibitory action of HepG2 hepatoma cell proliferation;
Fig. 5 to show in crocodile blood polysaccharide component ABPSI to the inhibitory action of BEL-7402 cell migration;
Fig. 6 to show in crocodile blood polysaccharide component ABPSII to the inhibitory action of BEL-7402 cell migration;
Fig. 7 to show in crocodile blood polysaccharide component ABPSIII to the inhibitory action of BEL-7402 cell migration;
Fig. 8 to show in crocodile blood polysaccharide component ABPSI to the inhibitory action of SMMC-7721 cell migration;
Fig. 9 to show in crocodile blood polysaccharide component ABPSII to the inhibitory action of SMMC-7721 cell migration;
Figure 10 to show in crocodile blood polysaccharide component ABPSIII to the inhibitory action of SMMC-7721 cell migration;
Figure 11 to show in crocodile blood polysaccharide component ABPSI to the inhibitory action of HepG2 cell migration;
Figure 12 to show in crocodile blood polysaccharide component ABPSII to the inhibitory action of HepG2 cell migration;
Figure 13 to show in crocodile blood polysaccharide component ABPSIII to the inhibitory action of HepG2 cell migration.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described, it should be noted that the technical scheme in the embodiment of the present invention, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain, all belongs to the scope of protection of the invention.
Wherein, in embodiment, test material used and source thereof comprise:
1. cell line
BEL-7402 (human liver cancer cell): purchased from Nanjing KaiJi Biology Science Development Co., Ltd;
SMMC-7721 (human liver cancer cell): purchased from Nanjing KaiJi Biology Science Development Co., Ltd;
HepG2 (human liver cancer cell): purchased from Nanjing KaiJi Biology Science Development Co., Ltd;
Experimental agents and reagent
Crocodile blood lyophilized powder: Powdered, by Beijing, Bai Shengkang bio tech ltd provides.
Tetramethyl azo azoles salt (MTT): Shanghai Sheng Gong biological engineering company limited, article No.: TB0799-1g, lot number: XP1008B3012J, rank: analytical pure, specification 1g.
Dimethyl sulfoxide (DMSO): purchased from Shanghai Sheng Gong biological engineering company limited, article No.: DN3039A, lot number: SJ0731S4013J, rank: analytical pure, specification 500mL, for the crystallization of mtt assay dissolve purple.
Dimethyl sulfoxide (DMSO) cell culture level: purchased from Beijing Suo Laibao Science and Technology Ltd., article No.: D8370-500, lot number: 2P005970, rank: cell culture level, specification 500mL, for cell cryopreservation, and Pharmaceutical formulations.
RPMI1640 culture medium: purchased from Thermo Fisher Scientific Inc., lot number: NYG0920, specification: 500mL.
Trypsin: purchased from Shanghai Sheng Gong biological engineering company limited, article No.: T0458-10, lot number: 0301C314, rank: USP, specification: 10g.
3. key instrument
AKTA system: GE company, model: UPC-900
Biohazard Safety Equipment: the safe thing of Spain reaches (Telstar), model: BioIIA.
CO
2incubator: Shi Doukai instrument and equipment (Shanghai) Co., Ltd., model: STIKIL-161HI.
Inverted phase contrast microscope: producer: Olympus, model: CKX41.
Shimadzu UV-1800 ultraviolet spectrophotometer: Shimadzu business administration (China) company limited, model: UV-1800;
Electronic balance: Shanghai Ao Haosi Instrument Ltd., model: AR2202CN;
Tabletop refrigerated centrifuge: Thermo Fischer Scient Inc., model: ST16R;
Ultrasonic cell-break machine: Sai Mo flies generation that (ThermoFisher), model: SCIENTZ-IID;
Rotary Evaporators: Shanghai Yarong Biochemical Instrument Plant, model: RE-5205;
4 DEG C of refrigerators: Qingdao HaiEr Co., Ltd, model: BCD-192KJ;
Vortex vortex mixer: its woods Bel instrument manufacturing company limited of Haimen City, model: QZ-861.
4. the compound method of agents useful for same comprises:
Tetramethyl azo azoles salt (MTT) liquid storage: get MTT powder 1.0g, be dissolved in 200ml phosphate buffer (PBS), final concentration is 5mg/ml, and with 0.22 μm of membrane filtration to remove the antibacterial in solution, subpackage ,-20 DEG C keep in Dark Place.Wherein, solution preparation and use all should operate in sterile biological safety cabinet.
Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
The preparation method of embodiment 1 crocodile blood polysaccharide
1, the preparation of crocodile blood polysaccharide crude extract
Take crocodile blood lyophilized powder 4.0g, add corresponding distilled water (ddH2O) by solid-liquid ratio 1:20, in ultrasonic cell-break machine, broken 5min; 4600rpm/min in 4 DEG C of centrifuges, centrifugal 10min, gets supernatant, repeat 3 times, merge supernatant, add the dehydrated alcohol left undisturbed overnight of 3 times of volumes, 4600rpm/min, after centrifugal 10min, abandoning supernatant, precipitates with appropriate washes of absolute alcohol 3 times, centrifugal abandon supernatant after, powdered with appropriate ether piping and druming, obtain crocodile blood polysaccharide crude extract.
2, the separation and purification of crocodile blood polysaccharide different component
According to the feature of protein degeneration in chloroform equal solvent, after chloroform and n-butyl alcohol are pressed 4:1 volume mixture, be placed in brown bottle and preserve.Crocodile blood polysaccharide crude extract is dissolved in ddH2O, in polysaccharide solution, adds chloroform-butanol solution, after mixing by 1:4, thermal agitation 20min, the denatured protein in centrifugal rear removing intermediate layer, repeats, for several times to intermediate layer is separated out without protein, then to separate out lower floor's polysaccharide solution, be placed in bag filter, dialysis 72h, constantly changes water, to remove the small molecular weight impurities such as oligosaccharide, then concentrate, final lyophilization is polysaccharide powder.
Crocodile blood polysaccharide, after Deproteinization, remove impurity, is mixed with polysaccharide solution with ddH2O by polysaccharide, uses separation and purification system (AKTA system) to cross SephadexG-200 sephadex column and is separated further, use ddH before loading
2first O balances pillar, and after loading pin loading, system uses ddH automatically
2o carries out the eluting of polysaccharide, and the elution volume place that system does not coexist different according to molecular size range goes out peak, collects out the polysaccharide solution in the corresponding eluting pipe in peak position, and merges the polysaccharide under unified peak type.
As shown in Figure 1, summit is there is at 5.6,11.4,17.3 elution volume places respectively in the crude extract of crocodile blood polysaccharide after SephadexG-200 molecular sieving purification, thus be separated the polysaccharide component obtaining three kinds of different molecular weight sizes, be designated as respectively: ABPSI, ABPSII, ABPSIII.
The different component of embodiment 2 crocodile blood polysaccharide is on the impact of human hepatoma cell proliferation
Experimental technique is MTT colorimetry: Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that the crystallization of first a ceremonial jade-ladle, used in libation is formed is directly proportional to cell number.The method has been widely used in Activity determination, large-scale screening anti-tumor medicine, cell toxicity test and the tumor radiosensitivity mensuration etc. of some bioactie agents.
Method step is as follows:
(1) cell recovery and cultivation
Frozen human liver cancer cell is taken out from liquid nitrogen, drops in 37 DEG C of water-baths immediately and cell is melted.In Biohazard Safety Equipment, cell suspension is drawn onto in the centrifuge tube that appropriate culture medium is housed, centrifugal 5 minutes of 800rpm/min; Abandon supernatant, with 1mL culture medium suspension cell, be drawn onto in the Tissue Culture Dish that appropriate culture medium is housed, cell be placed in 37 DEG C, 5%CO
2, saturated humidity condition under cultivate.Reach when 80%-90% contact converges until cell and go down to posterity, 0.2% trypsinization becomes single cell suspension, centrifugal 5 minutes of 800rpm/min; Abandon supernatant, add 1-2mL culture medium suspension cell, cell is passed to 2-3 and be equipped with in the culture dish of appropriate culture medium, continue to cultivate.
(2) mtt assay measures the inhibitory action of medicine cell growth
By being in the attached cell of exponential phase after trypsinization, be dispersed into individual cells, and make it be suspended in corresponding cell culture medium.Cell is inoculated on 96 well culture plates, every hole 100 μ L cell suspension, 3000cells/ hole.Above-mentioned 96 well culture plates are placed in 37 DEG C, and in carbon dioxide (5%) incubator, incubated overnight 24 hours, makes cell attachment.
After 24 hours later cell are completely adherent, discard culture fluid, test group adds the cell culture fluid of ABPSI, ABPSII, ABPSIII containing variable concentrations respectively, and arranges and do not add medicine and only add the control wells of relative medicine solvent (crocodile blood polysaccharide solvent is ddH
2o), arrange simultaneously and only add the not celliferous zeroing hole of culture medium.Often group establishes 6 parallel holes, then 96 orifice plates is placed in 37 DEG C, cultivates 48 hours in carbon dioxide (5%) incubator.
After 48 hours, every hole adds 25 μ LMTT (concentration is 5mg/mL), continues to hatch 4h.Then by culture fluid sucking-off gently, every hole adds 150 μ LDMSO and makes dissolution with solvents, measures the absorbance at 570nm place after dissolving by microplate reader.
(3) data analysis
Calculate survival rate and the suppression ratio of cell, and calculate the half-inhibition concentration IC50 of medicine to cell.Survival rate %=(experimental group OD value-return to zero hole OD value)/(negative model group OD value-return to zero hole OD value) × 100%; Suppression ratio %=(1-survival rate) × 100%.The calculating of IC50 value is carried out with GraphpadPrism5 software; Measurement result is in table 1.
Table 1 crocodile blood polysaccharide different component is to the inhibitory action (IC50 value) of hepatoma carcinoma cell
| Group | BEL-7402 | SMMC-7721 | HepG2 |
| ABPSI | 1.419 | 1.358 | 1.941 |
| ABPSII | 1.415 | 1.024 | 3.993 |
| ABPSIII | 1.880 | 1.581 | 1.078 |
As can be seen from Table 1, when low concentration, crocodile blood polysaccharide has the effect of obvious Inhibit proliferaton to human liver cancer cell.
As in Figure 2-4, for the crocodile blood polysaccharide medicine of the variable concentrations different component of detection in embodiment 2 is to the inhibitory action curve chart of the growth of multiple different human liver cancer cell, can show that from figure concentration presents obvious inhibitory action with to the growth of human liver cancer cell when low concentration, along with the increase of crocodile blood polysaccharide concentration, the inhibitory action of human liver cancer cell is strengthened gradually, and presents obvious concentration dependent.
The impact that the different component of embodiment 3 crocodile blood polysaccharide moves human liver cancer cell
Method step is as follows:
(1) cell recovery and cultivation
Frozen human liver cancer cell is taken out from liquid nitrogen, drops in 37 DEG C of water-baths immediately and cell is melted.In Biohazard Safety Equipment, cell suspension is drawn onto in the centrifuge tube that appropriate culture medium is housed, centrifugal 5 minutes of 800rpm/min; Abandon supernatant, with 1mL culture medium suspension cell, be drawn onto in the Tissue Culture Dish that appropriate culture medium is housed, cell be placed in 37 DEG C, 5%CO
2, saturated humidity condition under cultivate.Reach when 80%-90% contact converges until cell and go down to posterity, 0.2% trypsinization becomes single cell suspension, centrifugal 5 minutes of 800rpm/min; Abandon supernatant, add 1-2mL culture medium suspension cell, cell is passed to 2-3 and be equipped with in the culture dish of appropriate culture medium, continue to cultivate.
(2) plating cells
First use marker pen and ruler at the bottom of the plate of 24 orifice plates, evenly must draw horizontal line, cross via hole, two, every hole horizontal line, spacing 0.2-0.5cm.By being in the attached cell of exponential phase after trypsinization, be dispersed into individual cells, and make it be suspended in corresponding cell culture medium.Cell is inoculated on 24 well culture plates, every hole 500 μ L cell suspension, 500000cells/ hole.Above-mentioned 24 well culture plates are placed in 37 DEG C, and in carbon dioxide (5%) incubator, incubated overnight 24 hours, makes cell attachment.
Until 24 hour cells are completely adherent cover with after, the horizontal line cut hung down at the bottom of as plate with 20 μ L rifle heads, rifle head wants vertical, can not tilt.After PBS cleans 3 times, test group adds the cell non-serum culture medium of ABPSI, ABPSII, ABPSIII containing variable concentrations respectively, and blank group adds the serum-free medium of equal volume.Observe and Taking Pictures recording under inverted fluorescence microscope when 0h, 24h, 48h respectively.
(3) data analysis
The calculating of inhibition of metastasis rate is carried out with GraphpadPrism5 software.
(image in Fig. 5-13 is Y mobility-X time graph as shown in figures 5-13, represent that the dosage of 0,1,2 μm of ol, tri-variable concentrations and administration time are on the impact of mobility respectively), illustrate the inhibitory action of three kinds of crocodile blood polysaccharide to three kinds of different human liver cancer cell travel motions, after showing effect 48h in diagram, three kinds of crocodile blood polysaccharide all have the effect of suppression fucosylation in various degree.
Wherein, as illustrated in figs. 5-7, be the inhibition of metastasis rates of three kinds of crocodile blood polysaccharide components to the effect of BEI-7402 cellular migration inhibition, can as seen from the figure, polysaccharide component concentration is higher, more obvious to the suppression migration of cancer cell.
As seen in figs. 8-10, three kinds of crocodile blood polysaccharide components, can as seen from the figure to the inhibition of metastasis rate of SMMC-7721 cellular migration inhibition effect, and polysaccharide component concentration is higher, more obvious to the suppression migration of cancer cell.
As figs 11-13, three kinds of crocodile blood polysaccharide components, can as seen from the figure to the inhibition of metastasis rate of HepG2 cellular migration inhibition effect, and polysaccharide component concentration is higher, more obvious to the suppression migration of cancer cell.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. crocodile blood polysaccharide is preparing the application in cancer treatment drug.
2. application according to claim 1, is characterized in that: the application in the medicine suppressing liver cancer growth or transfer is being prepared in described application.
3. application according to claim 1, is characterized in that: described crocodile blood polysaccharide is the mixture containing at least one polysaccharide component of gained after separation and purification from crocodile blood blood powder.
4. application according to claim 1, is characterized in that: described crocodile blood polysaccharide comprises 1 ~ 3 kind of polysaccharide component.
5. the application according to any one of claim 1-4, is characterized in that: the described pharmaceutical pack of Hepatoma therapy is containing crocodile blood polysaccharide and pharmaceutically acceptable acid, alkali, salt, hydrate or ester and other adjuvants.
6. application according to claim 5, is characterized in that: described medicine can be prepared at least one in tablet, capsule, oral liquid, emulsion and injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109512839A (en) * | 2019-01-09 | 2019-03-26 | 百生康（北京）健康产业科技有限公司 | A kind of crocodile Blood piece and its processing technology with anti-tumor function |
| CN112335767A (en) * | 2019-08-08 | 2021-02-09 | 盐城泰鳄湖生态公园有限公司 | Method for preparing anticancer candy from crocodile blood |
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| CN1634147A (en) * | 2004-10-19 | 2005-07-06 | 中山大学 | Application of crocodile blood in process for preparing anticancer antivirus and immunity improving medicine and health caring food |
| CN102106872A (en) * | 2010-03-26 | 2011-06-29 | 北京洪源澳达生物技术发展有限公司 | Crocodile blood freeze-dried powder, and preparation method and application thereof |
| CN104644794A (en) * | 2015-01-23 | 2015-05-27 | 广州善康生物科技有限公司 | Drug for treating lumps |
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2015
- 2015-10-26 CN CN201510702628.0A patent/CN105147720B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634147A (en) * | 2004-10-19 | 2005-07-06 | 中山大学 | Application of crocodile blood in process for preparing anticancer antivirus and immunity improving medicine and health caring food |
| CN102106872A (en) * | 2010-03-26 | 2011-06-29 | 北京洪源澳达生物技术发展有限公司 | Crocodile blood freeze-dried powder, and preparation method and application thereof |
| CN104644794A (en) * | 2015-01-23 | 2015-05-27 | 广州善康生物科技有限公司 | Drug for treating lumps |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109512839A (en) * | 2019-01-09 | 2019-03-26 | 百生康（北京）健康产业科技有限公司 | A kind of crocodile Blood piece and its processing technology with anti-tumor function |
| CN112335767A (en) * | 2019-08-08 | 2021-02-09 | 盐城泰鳄湖生态公园有限公司 | Method for preparing anticancer candy from crocodile blood |
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