CN105194645A - Application of bullacta exarata polypeptides in resisting prostatic cancer - Google Patents
Application of bullacta exarata polypeptides in resisting prostatic cancer Download PDFInfo
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Abstract
The invention relates to application of bullacta exarata polypeptides in resisting prostatic cancer, in particular to application of bullacta exarata polypeptides in medicine resisting prostatic cancer DU-145 cells. A bullacta exarata polypeptide crude extract is obtained through a trypsin enzymolysis method, all bullacta exarata polypeptide components are obtained through an ultrafiltration method, a gel column separation method, a reverse-phase high performance liquid chromatogram technology and other separation and purification means, the inhibiting capacity of each separated component on proliferation of prostatic cancer DU-145 cells is studied, and the study shows that all the components can remarkably inhibit proliferation of prostatic cancer DU-145 cells. The raw material cost is low, extraction conditions are mild, extracted bullacta exarata polypeptides can remarkably inhibit proliferation of prostatic cancer DU-145 cells, and theoretical bases are provided for developing prostatic cancer resisting medicine with bullacta exarata polypeptides as raw materials.
Description
Technical field
The present invention relates to the application of mud snail enzymolysis polypeptide, be specifically related to the application of mud snail polypeptide in anti-prostate cancer.
Background technology
Mud snail (Bullactaexarata), be commonly called as " Bullactaexarata (Philippi) ", " yellow mud spiral shell ", " prunus mume (sieb.) sieb.et zucc. spiral shell " etc., belong to Mollusca, Gastropoda, Opisthobranchia, head Parapet order, A Di spiral shell section, mud snail genus, for the kind of west bank, Pacific Ocean sea water and salt-fresh water special product, be made up of shell and software two parts, containing compositions such as rich in protein, calcium, phosphorus, ferrum and vitamin, peptide material enriches, being distributed widely in the Changjiang river of the East Sea and Huanghai Sea both sides in China, is a kind of common aquatic products.
Malignant tumor is the disease of a class serious threat human health and life, and sickness rate is more and more higher, and the problems such as though current existing chemotherapeutics has certain curative effect to most of tumor, poor selectivity, toxicity are large, drug resistance still clearly.Therefore, searching is efficient, low toxicity, special strong antitumor drug are still imperative.Polypeptide because its molecular weight is little, non-immunogenicity, activity is high, side effect is little, especially also has good inhibit activities to the tumor cell line of multi-drug resistance.Marine organisms polypeptide is a key areas of current marine biomaterial research, some biologically active polypeptides are often present in protein with inactive state, these protein are through the hydrolysis of protease, the bioactive peptide be hidden in wherein is discharged, has likely given play to biological activity more more than protein.Such as: as Deng Wei etc. finds that the growth in vitro of phycocyanin enzymolysis polypeptide to HeLa Cells strain has obvious inhibitory action; The Mytilus edulis polypeptide of the enzymolysis Mytilus edulis gained such as Yang Yongfang has specific inhibited proliferation to Human Prostate Cancer PC-3 Cell Line and DU-145 cell, and is better than the inhibited proliferation to PC-3 cell to the inhibitory action of DU-145 cell; Yao is as forever waited research Tegillarca granosa polypeptide at 0.25-1.0gL
-1in scope, polypeptide significantly can suppress the propagation of lung cell A549 and Ketr-3 cell, simultaneously can also the synthesis of T suppression cell protein, and in obvious dose dependent.
Carcinoma of prostate is male's malignant tumor occurred frequently, and its mortality rate is in the second of cancer mortality, is only second to pulmonary carcinoma.In recent years, along with the continuous lifting of the change of people life style, the aging of population and diagnostic level, the sickness rate that I crosses carcinoma of prostate is obviously in rising trend.Carcinoma of prostate DU-145 cell is separated from carcinoma of prostate brain metastes tumor, and differentiation degree is low, is the prostate gland cancer cell of Androgen-dependent, has powerful metastatic potential, lack the expression of endogenic androgen receptor.DU-145 cell completely adherent time is generally in 24h, has stronger invasion and attack and angiogenesis promoting ability.The inhibitory action of research mud snail enzymolysis polypeptide many carcinoma of prostate DU-145 cell, significant to the theoretical research of carcinoma of prostate medicine.
Summary of the invention
Technical problem to be solved by this invention obtains on the basis of mud snail polypeptide at enzymolysis mud snail tissue, provides the application of a kind of mud snail polypeptide in antiprostate cancer, especially anti-prostate cancer DU-145 cell.
The present invention solves the problems of the technologies described above adopted technical scheme: the application of a kind of mud snail polypeptide in anti-prostate cancer DU-145 cell drug, carcinoma of prostate DU-145 cell is separated from carcinoma of prostate brain metastes tumor, differentiation degree is low, for the prostate gland cancer cell of Androgen-dependent, there is powerful metastatic potential, lack the expression of endogenic androgen receptor, the specific implementation form of its Chinese medicine can be tablet, capsule, granule or pill etc.
Above-mentioned mud snail polypeptide is obtained by following steps: new fresh snail is cleaned and shelled, get mud snail tissue mashing, add distilled water homogenate, solid-liquid ratio is 1:(3 ~ 4), the pH value of homogenate is regulated to be 8 ~ 9 after homogenate, add the trypsin of 0.4 ~ 0.5% homogenate volume, 40 ~ 50 DEG C of insulation hydrolysis 7 ~ 9h, latter 95 ~ 100 DEG C are hydrolyzed, 10 ~ 15min carries out enzyme denaturing, at 4 DEG C, hydrolyzed solution is in the centrifugal 15 ~ 20min of 9500 ~ 10000r/min, get supernatant concentration dry enzymolysis crude extract, enzymolysis crude extract is dissolved in distilled water, separation and purification obtains each mud snail polypeptide fractions.
The concentration of above-mentioned enzymolysis crude extract, when 5 ~ 25mg/ml, be 36.9 ~ 80.7%, and the concentration of Proliferation Ability index and enzymolysis crude extract is proportionate to DU-145 cell inhibitory effect index.
Above-mentioned purification procedures is ultrafiltration or is followed successively by ultrafiltration and gel chromatography or is followed successively by ultrafiltration, gel chromatography and reversed-phase high-performance liquid chromatography.
Further, 10kd, 5kd and 3kd ultrafilter membrane is used in described ultrafiltration step, obtaining molecular weight is respectively the component A1 of 10 ~ 5kd, molecular weight is the component A3 that the component A2 of 5 ~ 3kd and molecular weight are less than 3kd, proliferation inhibition rate after 20 ~ 25mg/ml component A3 acts on DU-145 cell 36h is 77 ~ 78%, and the concentration of Proliferation Ability index and component A3 is proportionate.
Further again, in described gel chromatography, eluting is carried out to component A3, obtain H1 component, H2 component and H3 component respectively, Proliferation Ability index after wherein 5 ~ 20mg/ml component H2 acts on DU-145 cell 24h is for being 44.1 ~ 81.6%, and the concentration of Proliferation Ability index and component H2 is proportionate; Gel chromatography condition: select SephadexG-25 gel chromatography, with deionized water balance after dress post, concentration is 50mg/mL, loading 4mL, speed is 3ml/min, and mobile phase is ultra-pure water, 280nm ultraviolet detection.
Further more again, reversed-phase high-performance liquid chromatography is adopted to be separated described H2 component, be separated and obtain component F1 and F2, after wherein 2.5 ~ 3mg/ml component F1 and component F2 acts on DU-145 cell 24h, 31 ~ 33.72% and 40 ~ 42.04% are respectively to the proliferation inhibition rate of DU-145 cell; Reversed-phase high-performance liquid chromatography condition: select ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Mobile phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is that 50ul ultraviolet detection wavelength is respectively 214nm, 280nm.
The aminoacid sequence of said components F1 is as described in SEQIDNO.1, and the aminoacid sequence of component F2 is as described in SEQIDNO.2.
Compared with prior art, the invention has the advantages that: carcinoma of prostate is a kind of malignant tumor having a strong impact on men's health, the present invention obtains enzymolysis crude extract by trypsin digestion mud snail tissue, then obtained the various ingredients of mud snail polypeptide by separation and purification means, study the impact of each component on carcinoma of prostate DU-145 cell.The mud snail adopted in the present invention is a kind of low value shellfish, the cost of raw material is low, and the extracting method technique of mud snail polypeptide is simple, reaction temperature and, extract each component of mud snail polypeptide obtained remarkable to the proliferation inhibiting effect of carcinoma of prostate DU-145 cell, wherein component H2 is to the IC of DU-145 cell
50for 1.32mg/ml, for exploitation provides theoretical foundation with the antiprostate cancer that mud snail polypeptide is raw material.
Accompanying drawing explanation
Fig. 1 be in embodiment 4 mud snail enzymolysis crude extract to the suppression ratio of carcinoma of prostate DU-145 cell;
Fig. 2 be in embodiment 4 membrance separation component to the suppression ratio of DU-145 cell;
Fig. 3 be in embodiment 4 A3 component through SephadexG-25 column chromatography curve;
Fig. 4 be in embodiment 4 gel chromatography component to the broken line graph of the suppression ratio of DU-145 cell;
Fig. 5 be in embodiment 4 gel chromatography component to the block diagram of the suppression ratio of DU-145 cell;
Fig. 6 is the RP-HPLC collection of illustrative plates of H2 component in embodiment 4;
The component of Fig. 7 reversed-phase high-performance liquid chromatography purification is to the suppression ratio of DU-145 cell.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of mud snail polypeptide
(1.1) Feedstock treating
Get new fresh snail, mud snail is shelled, to drain rear homogenate for subsequent use.
(1.2) mud snail enzymolysis process
With high-speed tissue mashing machine by the homogenate of mud snail tissue mashing, precision takes homogenate, with the hydrochloric acid solution of 0.1mol/L and the NaOH solution adjust pH of 0.1mol/L, add trypsin digestion a few hours, enzymatic hydrolysis condition is hydrolysis temperature 45 DEG C, and pH is 8.7, solid-liquid ratio 1:4, enzymolysis time 8h, enzyme concentration 0.48%.Enzyme denaturing 15min at 100 DEG C, centrifugal 15min (10000r/min) at 4 DEG C, it is for subsequent use to get supernatant concentration.
The cultivation of embodiment 2:DU-145 cell and going down to posterity
(2.1) cell recovery
The DU-145 cell strain deposited is taken out from liquid nitrogen container, put into 37 DEG C of thermostat water baths fast to melt, after melting, enter aseptic operating room operate, cell strain is sucked centrifuge tube by the suction pipe good with sterilizing, add F12 nutritional solution or the RPMI1640 nutritional solution of 2mL, the centrifugal 10min of 1000rpm, removes supernatant, add the nutritional solution of 4ml, piping and druming makes cell become individual cells repeatedly.Then with suction pipe, cell is drawn in the culture bottle of 2 25mL uniformly, puts into 5%CO
2, the constant incubator of 37 DEG C cultivates, change liquid next day and outwell not adherent dead cell.
(2.2) cell culture
Human Prostate Cancer Cells DU-145 is inoculated in the F12 and 1640 nutritional solutions containing 10% hyclone (volume fraction) FBS and dual anti-(benzylpenicillin 100IU/mL, streptomycin 100IU/mL) respectively, is positioned over 37 DEG C, 5%CO
2constant incubator in cultivate, cell attachment grow, within every 1 day, change liquid once, go down to posterity when cell covers with about 80% of culture bottle.Go down to posterity according to the ratio of 1: 2.Collect exponential phase cell to test.
(2.3) passage
First the culture bottle covering with cell is taken out from constant incubator and be put on aseptic operating platform.When going down to posterity, first outwell the nutritional solution in bottle, remove the dead cell of not adherent growth, with 0.25% trypsin/0.02%EDTA Digestive system mixture slaking, the time of different cell dissociation is different, and general digestion time is 3-5min; Basis of microscopic observation cell, when intercellular substance obviously becomes large and cell rounding brightens, illustrates that cell has digested complete, removes digestive enzyme liquid.In culture bottle, add the nutritional solution of about 2.5mL, repeatedly blow and beat the attached cell digested and make it to become individual cells, ordinary circumstance next bottle of cell passes 2 bottles, the cell gone down to posterity is placed in 37 DEG C, 5%CO
2incubator in cultivate.
(2.4) cell cryopreservation
Basis of microscopic observation, when cell grows to about 80% of culture bottle, select cellular morphology good carry out frozen, remove culture fluid, add Digestive system to digest cell, digestive enzyme is outwelled when Microscopic observation intercellular substance is obvious, in culture bottle, add the nutritional solution of about 3mL again, piping and druming makes it to become individual cells repeatedly, sucks in centrifuge tube, 1000rpm, centrifugal 10min, culture fluid is abandoned in careful suction, adds containing 10%DMSO hyclone 1mL, with suction pipe repeatedly blow and beat cell evenly after, be drawn in aseptic cryopreservation tube.After first putting into 4 DEG C of refrigerator 30min, then put into liquid nitrogen position of bottleneck 2h, finally put into liquid nitrogen bottle and carry out frozen.
: embodiment 3: the separation and purification of enzymolysis crude extract
First ultrafiltration is selected to be separated mud snail polypeptide enzymolysis crude extract, ultrafilter membrane selects 10kd respectively, 5kd, 3kd, be trapped into molecular weight 10-5kd, 5-3kd respectively and be less than 3kd tri-components, be made into the concentration of 20mg/mL after lyophilization respectively, measure the suppression ratio of each component to carcinoma of prostate DU145 with mtt assay.Select an active the highest component, select SephadexG-25 gel chromatography, with deionized water balance after dress post, concentration is 50mg/mL, applied sample amount is 4mL, speed is 3ml/min, and mobile phase is ultra-pure water, 280nm ultraviolet detection.Collect eluting peak lyophilization powdered, detect the proliferation inhibition rate to DU-145 cell, and curve plotting draws IC
50, efficient liquid phase chromatographic analysis is carried out in a large amount of collection lyophilizations the highest for activity.
By the water dissolution of mud snail sample 0.06%TFA good for lyophilization in the centrifuge tube of 0.6ml, at 12000rpm, centrifugal 10min gets supernatant.Efficient liquid-phase condition: select ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Mobile phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is 50ul, and ultraviolet detection wavelength is respectively 214nm, 280nm.
Embodiment 4:MTT method explores mud snail polypeptide anti-tumor activity
The cell of trophophase of taking the logarithm makes suspension, is seeded to 96 orifice plates, every hole 200 μ L, if 5 parallel holes, in 5%CO
2, 37 DEG C of adherent 16-48h, observe under inverted microscope, discard culture fluid, are dissolved in respectively in culture fluid by testing sample with variable concentrations simultaneously.Then add each hole respectively, establish the matched group not adding sample simultaneously, put 5%CO
2, hatch 36h in 37 DEG C of incubators, rinse 2 times with PBS, add the nutritional solution containing MTT, continue to cultivate 4h.Stop cultivating, carefully suck culture fluid in hole.Add DMSO, put low-speed oscillation 10min on shaking table, adopt enzyme-linked immunosorbent assay instrument to survey absorbance (OD value) in 490nm.Calculate cell inhibitory effect index (IR, Inhibitionrate), i.e. proliferation inhibition rate, by following formulae discovery:
(4.1) mud snail enzymolysis crude extract antitumor activity
Mud snail good for homogenate is carried out enzymolysis with trypsin, and centrifuging and taking supernatant carries out lyophilization, obtains mud snail polypeptide crude extract, adopts mtt assay to observe the impact of its induction human prostate cancer cell line DU 145 strain apoptosis in vitro.Result shows: along with the increase suppression ratio of peptide concentration increases gradually, when concentration is 5mg.mL
-1time, proliferation inhibition rate is 36.9%, when concentration is 20mg.mL
-1time, suppression ratio reaches 79.6%, and it is active that this illustrates that this polypeptide has certain anti-prostate cancer.As shown in table 1 and Fig. 1.
The suppression ratio (x ± s, n=5) of table 1 pair DU-145 cell
Note: * compared with the minimum cell inhibitory rate of each component, P<0.05; With mud snail polypeptide after cell incubation 36h to the IC of DU-145 cell inhibitory effect
50for 2.425mg/ml.
(4.2) the membrance separation component antitumor activity of mud snail enzymolysis polypeptide
Step obtains three components as described in Example 3, be molecular weight 10-5kd (A1) respectively, 5-3kd (A2), be less than 3kd (A3), respectively each component is carried out lyophilization, and the concentration being mixed with 20mg/mL carries out MTT experiment, after effect 36h, the suppression ratio of each component to DU-145 cell is respectively 49.8%, 65.7%, 78.3%.As shown in Figure 2, wherein A3 component is better than other two components to the inhibitory action effect of DU-145 cell.
(4.3) antitumor activity of gel chromatography component
After ultrafiltration, obtain three components, know that A3 component is best by 4.2 experiments, A3 component is collected in a large number, lyophilization is carried out after concentrated, select gel SephadexG-25 chromatography by the experiment condition eluting of embodiment 3, obtain three component H1, H2, H3, as shown in Figure 3.
Each peak component is made into respectively the concentration of 5.0mg/mL, 10.0mg/mL, 15.0mg/mL, 20.0mg/mL, the inhibited proliferation to carcinoma of prostate DU-145 tumor cell is detected respectively with mtt assay, after cultured cell 24h, the sample preparing concentration is added cell, blank group is set, by SPSS software to independent sample T check analysis, other concentration group and this group have there was no significant difference, and P<0.05 shows to there is significant difference with blank group.By table 2 curve plotting figure as shown in Figure 4,5, in Fig. 5, the post bar of each dosage group is followed successively by H1, H2 and H3 from left to right.Cross plot analysis can draw, 3 kinds of components have certain inhibition to DU-145 tumor cell, and present dose-effect relationship, wherein the anti-tumor activity of H2 is the highest, when 20.0mg/mL, can reach 81.6% to the suppression ratio of DU-145, inhibition is remarkable, as shown in table 2.
The each component of table 2 is to the proliferation inhibition rate of DU-145 cell
(n=3)
Note: ﹡ compared with the minimum cell inhibitory rate of each component, P<0.05
(4.4) antitumor activity of reversed-phase high-performance liquid chromatography purified components
Anti-tumor activity through the known H2 component of 4.3 experiment is best, is separated further, detects comparison, as shown in Figure 6 labelling respectively, namely obtain F1 and F2 respectively through high performance liquid chromatography at 214nm and 280nm ultraviolet wavelength H2 component.
The activity being separated the component H2 obtained through gel chromatography is the highest, and obtaining 2 components in concentration through HPLC separation is 3mg/mL, after effect 24h, is respectively 33.72% and 42.04%, and draws block diagram, see Fig. 7 to the proliferation inhibition rate of DU-145 cell.
As can be seen from Fig. 7, component F2 is to the inhibited proliferation of DU-145 tumor cell, be better than the inhibiting tumour cells effect of F1 component, F1, F2 component is carried out collecting and with PPSQ-31A, structure detection is carried out to it, the aminoacid sequence of F1 is as described in SEQIDNO.1 after testing, and the aminoacid sequence of F2 is as described in SEQIDNO.2.
The various embodiments described above experiment material used and experimental apparatus as follows:
Experiment material
New fresh snail collection is from Zhoushan Of Zhejiang Province offshore sea waters;
Papain; Trypsin Chemical Reagent Co., Ltd., Sinopharm Group);
Formaldehyde (37.0%-40.0%); Chemical Reagent Co., Ltd., Sinopharm Group;
Hydrochloric acid (36%-38%); Chemical Reagent Co., Ltd., Sinopharm Group's top grade is pure;
Sodium hydroxide; Wuxi City Jing Ke Chemical Co., Ltd. analytical pure;
Methyl thiazoly tetrazolium assay (MTT), SIGMA company of the U.S.;
F12 powder culture medium, SIGMA company of the U.S.;
RPMI1640 culture medium, Gibco company;
Dimethyl sulfoxide (DMSO), SIGMA company of the U.S.;
Acetonitrile, (CAN, FisherScientific company)
Trifluoroacetic acid, (TFA, Merck company)
Polybrene, (Polybrene, ShimadzuCorporation);
The amino acidity scale product of PTH-, (PTH-AA, ShimadzuCorporation);
Experimental apparatus
The automatic fraction collector of BSZ-40-LCD, Shanghai Qi Te Analytical Instrument Co., Ltd;
Agilent1260(Agilent.USA)
Ultrafiltration cup, ultrafilter membrane, rub fast science equipment company limited in Shanghai;
G-25 gel molecular sieves, Heng Xin bio tech ltd, Asia-Pacific, Beijing;
FD-1000 freezer dryer, Shanghai Ai Lang Instrument Ltd.;
CF16RXII High speed refrigerated centrifuge, Japanese HITACHI company;
UV1100 ultraviolet spectrophotometer, Shanghai Mei Puda company;
BSA124S type electronic balance, German SartoriousAG company;
VPWS-T-20L Superpure water machine, Hangzhou Yongjieda Cleaning Technology Co., Ltd;
ZHJH-C1209C type superclean bench, Shanghai Zhi Cheng analytical tool Manufacturing Co., Ltd;
Microplate reader, U.S. Bio-Rad Products;
Forma3111 type CO
2incubator, Thermo company of the U.S.;
Inverted microscope, Japanese OLYMPUS company;
PPSQ-31A, (ShimadzuCorporation, Japan);
PTFE filter membrane, (PTFEfilter, ShimadzuCorporation);
Glass fibre membrane, (GlassFiberDisk, ShimadzuCorporation);
Cell strain
Human prostata cancer DU-145 cell is purchased from Chinese Academy of Sciences's Shanghai cytobiology cell bank.
Claims (8)
1. the application of mud snail polypeptide in anti-prostate cancer DU-145 cell drug.
2. the application of mud snail polypeptide in anti-prostate cancer DU-145 cell drug as claimed in claim 1, it is characterized in that described mud snail polypeptide is obtained by following steps: new fresh snail is cleaned and shelled, get mud snail tissue mashing, add distilled water homogenate, solid-liquid ratio is 1:(3 ~ 4), the pH value of homogenate is regulated to be 8 ~ 9 after homogenate, add the trypsin of 0.4 ~ 0.5% homogenate quality, 40 ~ 50 DEG C of insulation hydrolysis 7 ~ 9h, latter 95 ~ 100 DEG C are hydrolyzed, 10 ~ 15min carries out enzyme denaturing, at 4 DEG C, hydrolyzed solution is in the centrifugal 15 ~ 20min of 9500 ~ 10000r/min, get supernatant concentration dry enzymolysis crude extract, enzymolysis crude extract is dissolved in distilled water, separation and purification obtains each mud snail polypeptide fractions.
3. the application of mud snail polypeptide in anti-prostate cancer DU-145 cell drug as claimed in claim 2, it is characterized in that: the concentration of described enzymolysis crude extract is when 5 ~ 25mg/ml, be 36.9 ~ 80.7% to DU-145 cell inhibitory effect index, and the concentration of Proliferation Ability index and enzymolysis crude extract is proportionate.
4. the application of mud snail polypeptide in anti-prostate cancer DU-145 cell drug as claimed in claim 2 or claim 3, is characterized in that: described purification procedures is ultrafiltration or is followed successively by ultrafiltration and gel chromatography or is followed successively by ultrafiltration, gel chromatography and reversed-phase high-performance liquid chromatography.
5. the application of mud snail polypeptide in anti-row adenocarcinoma DU-145 cell drug as claimed in claim 4, it is characterized in that: in described ultrafiltration step, use 10kd, 5kd and 3kd ultrafilter membrane, obtaining molecular weight is respectively the component A1 of 10 ~ 5kd, molecular weight is the component A3 that the component A2 of 5 ~ 3kd and molecular weight are less than 3kd, proliferation inhibition rate after 20 ~ 25mg/ml component A3 acts on DU-145 cell 36h is 77 ~ 78%, and the concentration of Proliferation Ability index and component A3 is proportionate.
6. the application of mud snail polypeptide in anti-row adenocarcinoma DU-145 cell drug as claimed in claim 5, it is characterized in that: in described gel chromatography, eluting is carried out to component A3, obtain H1 component, H2 component and H3 component respectively, Proliferation Ability index after wherein 5 ~ 20mg/ml component H2 acts on DU-145 cell 24h is for being 44.1 ~ 81.6%, and the concentration of Proliferation Ability index and component H2 is proportionate;
Gel chromatography condition: select SephadexG-25 gel chromatography, with deionized water balance after dress post, concentration is 50mg/mL, applied sample amount is 4mL, speed is 3ml/min, and mobile phase is ultra-pure water, 280nm ultraviolet detection.
7. the application of mud snail polypeptide in anti-row adenocarcinoma DU-145 cell drug as claimed in claim 6, it is characterized in that: adopt reversed-phase high-performance liquid chromatography to be separated described H2 component, be separated and obtain component F1 and F2, after wherein 2.5 ~ 3mg/ml component F1 and component F2 acts on DU-145 cell 24h, 31 ~ 33.72% and 40 ~ 42.04% are respectively to the proliferation inhibition rate of DU-145 cell;
Reversed-phase high-performance liquid chromatography condition: select ZorbaxSB-C18 (4.6 × 250,5um); Column temperature is 20 DEG C; Mobile phase is 1%TFA and acetonitrile; Gradient elution: from terminate to 30min, acetonitrile concentration changes to 40% from 0, and elution speed is 1.0mL/min; Sampling volume is 50ul; Ultraviolet detection wavelength is respectively 214nm, 280nm.
8. the application of mud snail polypeptide in anti-row adenocarcinoma DU-145 cell drug as claimed in claim 7, is characterized in that: the aminoacid sequence of described component F1 is as described in SEQIDNO.1, and the aminoacid sequence of component F2 is as described in SEQIDNO.2.
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| CN111534487A (en) * | 2020-06-22 | 2020-08-14 | 青岛思拓新源细胞医学有限公司 | Promoter for promoting osteogenic differentiation of mesenchymal stem cells |
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Cited By (2)
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| CN111534487A (en) * | 2020-06-22 | 2020-08-14 | 青岛思拓新源细胞医学有限公司 | Promoter for promoting osteogenic differentiation of mesenchymal stem cells |
| CN111534487B (en) * | 2020-06-22 | 2021-05-14 | 孙德强 | Promoter for promoting osteogenic differentiation of mesenchymal stem cells |
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