CN101962399A - Method for extracting natural antitumor active protein and polypeptide from chickpea bean - Google Patents
Method for extracting natural antitumor active protein and polypeptide from chickpea bean Download PDFInfo
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- CN101962399A CN101962399A CN 201010502809 CN201010502809A CN101962399A CN 101962399 A CN101962399 A CN 101962399A CN 201010502809 CN201010502809 CN 201010502809 CN 201010502809 A CN201010502809 A CN 201010502809A CN 101962399 A CN101962399 A CN 101962399A
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Abstract
The invention relates to a method for extracting natural antitumor active protein and polypeptide from chickpea bean. In the method, protein and polypeptide capable of inhibiting the activity of colonic cancer cells can be obtained by the steps of extracting phosphate buffer solution, precipitating ammonium sulfate, desalting, centrifuging and the like. The method is a basic research on bioactive substances in chickpea which is a Uighur medicinal food dual-purpose plant, and provides a basis for developing related products. The method has the advantages that the extraction process is simple, and the protein and the polypeptide can not be deactivated easily. The prepared polypeptide has the advantages of high biological activity, no toxicity and good thermal stability. The method can be easy for production in a large scale, reduces the cost of the extraction process, and is an ideal method for extracting and preparing bioactive protein and polypeptide. The protein and the polypeptide prepared by the method can be used for preparing functional foods or drug additives.
Description
Technical field
The present invention relates to a kind of method of from the garbanzo bean cotyledon, extracting natural antitumor active protein and polypeptide.
Background technology
Tumour has become the second largest killer who is only second to cardiovascular and cerebrovascular diseases at present, although the oncotherapy pattern has obtained remarkable progress at present, tumour is still all very high a kind of disease of M ﹠ M in the global range.In the U.S., tumour is to cause crowd's main causes of death below 85 years old.And many tumours, such as skin carcinoma, prostate cancer, mammary cancer, the sickness rate of kidney etc. is still continuing rising.Human health in the tumour serious threat, has become one of important social concern that countries in the world face.Seeking antitumor drug efficient, low toxicity is one of present worldwide research focus.
Yet, at present still not have a kind of anti-tumor active substance and carry out fairly large commercialization production, its reason may be that the medicine source problem is difficult to solve, and perhaps simultaneously also has certain toxic side effect having clear and definite anti-tumor activity, is difficult to directly use clinically.
To be that a class is natural be present in the organisms such as animal and plant and microorganism biologically active peptides (bioactive peptides), or animal and plant protein also can obtain through methods such as proteolysis and artificial chemosynthesis, biotechnology.Having found hundreds of kind peptide in the organism, is that body is finished the active requisite participant of various complex physiologic.All biomass cellss can both synthesize peptide material, and its functional activity also is subjected to the adjusting of polypeptide.In recent years, both at home and abroad the investigator attempts to obtain from crude substance and has the single-activity tumor protein p53, to overcome existing adverse drug reaction and drug resistance of tumor.Compare with traditional chemotherapy and radiation, natural antitumor active polypeptide biggest advantage is that relative molecular mass is little, activity is high, untoward reaction is little, is easy to the multipath absorption, is difficult for producing resistance.And they exist more the endogenous target spot, very easily penetrate tumour cell, improve immunne response, suppress tumor vessel and form, suppress characteristics such as tumor growth and transfer, become a new focus of oncotherapy research.
In decades, the exploration that people do not stop in field of antineoplastic medicaments has always obtained certain achievement.From natural resource, seek the specificity height, activity is strong, toxic side effects is little and uses wide anti-cancer agent research extensively to carry out, at present found that the antitumor drug of a lot of natural origins has better curative effect, clear and definite its mechanism of action, help developing its resulting structure and also improved, make it better to bring into play molecular targeted effect.Along with the continuous development of biotechnology, the antitumor drug research and development have welcome the new epoch, and this also provides optimistic prospect for the research and development of anti-tumor biological peptide.
Garbanzo (Cicer arietinumol/L L.) belongs to pulse family, pea family, the olecranon Macroptilium, growth history in 2500 is arranged in Xinjiang, is the Uygur medicine medicinal herbs most in use, and Uighur promise by name is carried recklessly, recorded in Ministry of Health of the People's Republic of China's " drug standard " Uygur medicine fascicle and " Uygur medicine will ", " it has the unusual body fluid of removing, opens the body fluid impatency, regulates effects such as body.Be used for asthenic body, hyposexuality, poor appetite, diseases such as skin pruritus and diabetes ".Though the garbanzo medication is with a long history, its functional factor is unclear, particularly the research blank of biologically active polypeptide compounds.
The object of the present invention is to provide a kind of method of from the garbanzo bean cotyledon, extracting natural antitumor active protein and polypeptide, this method after by phosphate buffer solution extraction, ammonium sulphate precipitation and desalination step purifying such as centrifugal obtain having the protein and the polypeptide of anti-tumor activity, be the biologically active substance fundamental research of Uygur medicine food dual purpose plant garbanzo, and develop corresponding product foundation is provided.This method preparation process is simple, is difficult for making protein and polypeptide inactivation again, the protein of preparation and polypeptide anti-tumor activity height, nontoxicity, Heat stability is good, being easy to enlarge scale production, reducing the purge process cost, is one of Perfected process of preparation biological activity protein and polypeptide.Protein that obtains by this method and polypeptide are as the purposes of preparation food, drug additive.
Summary of the invention
The object of the invention is, a kind of method of extracting natural antitumor active protein and polypeptide from the garbanzo bean cotyledon is provided, this method obtains the protein and the polypeptide of biologically active (as anti-tumor activity) by phosphate buffer solution extraction, ammonium sulfate precipitation, be the biologically active substance fundamental research of Uygur medicine food dual purpose plant garbanzo, and develop corresponding product foundation is provided.This method leaching process is simple, is difficult for making protein and polypeptide inactivation again, the protein of preparation and polypeptide anti-tumor activity height, nontoxicity, Heat stability is good, being easy to enlarge scale production, reducing production costs, is one of Perfected process of preparation biological activity protein and polypeptide.Protein that obtains by this method and polypeptide are as the purposes of preparation food, drug additive.
A kind of method of extracting natural antitumor active protein and polypeptide from the garbanzo bean cotyledon of the present invention follows these steps to carry out:
A, garbanzo cleaned remove foreign material, soak 1h-8h;
B, the garbanzo that step a is cleaned, soaks are put into the bean sprouts case and are germinateed, and temperature is 15-30 ℃, and water temperature 15-30 ℃, trickle time 3-10 second, trickle 2-6 hour at interval, germinating time 3-7 days;
Bud on c, the garbanzo bean sprouts that step b is germinateed and bean cotyledon separately and dry respectively;
D, the step c exsiccant garbanzo bean cotyledon that dries in the air is pulverized, cross the 10-60 mesh sieve after, carry out the cold soaking degreasing with normal hexane, the gained spent meal is standby;
E, with steps d spent meal liquid nitrogen grinding, stir to extract with phosphoric acid buffer again, the time is 1-5 hour, filters, and filtrate is removed precipitation with refrigerated centrifuge, it is standby to obtain supernatant liquor;
F, step e supernatant liquor is added anhydrous slufuric acid ammonia, fully place refrigerator after the stirring and dissolving and left standstill low-temperature centrifugation 24 hours, time 20min will precipitate with behind the water dissolution dialysis desalting, carry out low-temperature centrifugation again, time 20min, the precipitation drying can obtain anti-tumor active protein matter and polypeptide;
G, utilize the protein that the ordinary method sodium dodecyl sulfate-polyacrylamide gel electrophoresis determine to extract and the composition and the molecular weight thereof of polypeptide again, the activity that the protein that extracts and polypeptide suppress colon cancer cell is determined the protein of extraction and the anti-tumor activity of polypeptide.
Extraction among step e, f and the g, separate, purge process institute water is tri-distilled water.
Low-temperature centrifugation speed among step e, the f is 12000 rev/mins.
Concentration of phosphate buffer is 0.01-0.1mol/L among the step e,
pH=4.0-10.0。
Anhydrous slufuric acid ammonium saturation ratio is 30%-80% among the step f.
Garbanzo natural antitumor active protein that obtains by method of the present invention and polypeptide are as the purposes of the additive of preparation food or medicine.
Description of drawings
Fig. 1 is the 15%SDS-PAGE coomassie colored graph of garbanzo bean cotyledon active protein of the present invention and polypeptide, wherein: 0: the standard protein mark, Rat parathyroid hormone 1-34 (1-34) (4.1kDa), Trypsin inhibitor,Trasylol (6.5kDa), Rat parathyroid hormone 1-34 (1-84) (9.5kDa), N,O-Diacetylmuramidase (14.4kDa), trypsin inhibitor (20.0kDa), triosephosphate isomerase (27.0kDa), porcine pepsin (35.0kDa), ovalbumin (45.0kDa), bovine serum albumin (66.0kDa); No. 1 is embodiment 1 among the figure; No. 2 is embodiment 2; No. 3 is embodiment 3; No. 4 is embodiment 4; No. 5 is embodiment 5; No. 6 is embodiment 6.
Embodiment
The preparation of garbanzo bean cotyledon:
Outer foreign material are removed in the cleaning of 1kg garbanzo, soaked 1h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 15 ℃, 20 ℃ of water temperatures, 3 seconds trickle time, trickle 2 hours at interval, germinating time 4 days;
The bud and the bean cotyledon of 4 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 10 mesh sieves, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon powder liquid nitrogen grinding of 100 gram degreasings, is 0.1mol/L with concentration again, and the phosphoric acid buffer of pH=6.0 stirs for 4 ℃ in temperature and extracted 2 hours, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 30% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min will precipitate dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min, obtain water soluble protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
The preparation of garbanzo bean cotyledon:
Outer foreign material are removed in the cleaning of 1kg garbanzo, soaked 4h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 20 ℃, 15 ℃ of water temperatures, 5 seconds trickle time, trickle 6 hours at interval, germinating time 3 days;
The bud and the bean cotyledon of 3 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 40 mesh sieves, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon powder liquid nitrogen grinding of 100 gram degreasings, is 0.05mol/L with concentration again, and the phosphoric acid buffer of pH=7.0 stirs for 4 ℃ in temperature and extracted 1 hour, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 50% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min, precipitation, throw out is adopted dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min obtains water miscible protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
The preparation of garbanzo bean cotyledon:
Outer foreign material are removed in the cleaning of 1kg garbanzo, soaked 5h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 25 ℃, 25 ℃ of water temperatures, and 7 seconds trickle time, trickle 4 hours at interval germinateed 5 days;
The bud and the bean cotyledon of 5 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 60 mesh sieves, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon powder liquid nitrogen grinding of 100 gram degreasings, is 0.07mol/L with concentration again, and the phosphoric acid buffer of pH=5.0 stirs for 4 ℃ in temperature and extracted 3 hours, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 70% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min, precipitation, throw out is adopted dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min obtains water soluble protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
The preparation of garbanzo bean cotyledon:
Outer foreign material are removed in the cleaning of 1kg garbanzo, soaked 8h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 30 ℃, 30 ℃ of water temperatures, and 8 seconds trickle time, trickle 5 hours at interval germinateed 6 days;
The bud and the bean cotyledon of 6 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 50 mesh sieves, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon powder liquid nitrogen grinding of 100 gram degreasings, is 0.01mol/L with concentration again, and the phosphoric acid buffer of pH=9.0 stirs for 4 ℃ in temperature and extracted 5 hours, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 40% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min, precipitation, throw out is adopted dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min obtains water soluble protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
The preparation of garbanzo bean cotyledon:
Outer foreign material are removed in the cleaning of 1kg garbanzo, soaked 2h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 22 ℃, 25 ℃ of water temperatures, and 10 seconds trickle time, trickle 6 hours at interval germinateed 7 days;
The bud and the bean cotyledon of 7 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 30 mesh sieves, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon liquid nitrogen grinding of 100 gram degreasings, is 0.03mol/L with concentration again, and the phosphoric acid buffer of pH=4.0 stirs for 4 ℃ in temperature and extracted 4 hours, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 60% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min, precipitation, throw out is adopted dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min obtains water soluble protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
The preparation of garbanzo bean cotyledon:
Clear the 1kg garbanzo out outer foreign material, soak 6h;
The garbanzo of cleaning, soak is put into the bean sprouts case germinate, the temperature of bean sprouts case is 20 ℃, 20 ℃ of water temperatures, and 4 seconds trickle time, trickle 3 hours at interval germinateed 4 days;
The bud and the bean cotyledon of 4 days garbanzo of germination are separated, and dry respectively;
Exsiccant garbanzo bean cotyledon is pulverized, behind 20 mesh sieve, carried out the cold soaking degreasing with normal hexane excessively, the gained spent meal is standby;
The extraction of protein and polypeptide:
Getting the garbanzo bean cotyledon powder liquid nitrogen grinding of 100 gram degreasings, is 0.08mol/L with concentration again, and the phosphoric acid buffer of pH=10.0 stirs for 4 ℃ in temperature and extracted 3 hours, and extracting solution is used filtered through gauze earlier, removes precipitation with refrigerated centrifuge again, and it is standby to obtain supernatant liquor;
Separating of protein and polypeptide:
It is 80% that supernatant liquor is added anhydrous slufuric acid ammonia to its saturation ratio according to volume, fully putting into refrigerator after the stirring and dissolving left standstill 24 hours, low-temperature centrifugation, time 20min, precipitation, throw out is adopted dialysis desalting, with the protein after the desalination and polypeptide solution low-temperature centrifugation once more, time 20min obtains water soluble protein and polypeptide and water-insoluble protein and polypeptide, and cryodrying respectively, water-insoluble protein and polypeptide position are anti-tumor active protein matter and polypeptide.
Garbanzo bean cotyledon active protein that described method obtains and polypeptide are as the purposes of the additive of preparation food or medicine.
Embodiment 7
The garbanzo bean cotyledon active protein and the polypeptide that obtain by the method for the invention carry out determining of molecular weight ranges:
Reagent preparation and sample preparation:
The tetrabromophenol sulfonphthalein of the beta-mercaptoethanol of 10% sodium laurylsulfonate of Tutofusin tris-hydrogenchloride of the 0.5mol/L of sample buffer: 0.5mL (pH:6.8), 2mL, 1mL glycerine, 0.5mL, the water of 1mL, trace;
The processing of sample: sample solution and sample buffer equal-volume mix, and water proof boils 10min, centrifugal 10min;
Electrode buffer: 15.1 gram Tutofusin triss, 94 are restrained the 10% sodium laurylsulfonate dissolving of glycine, 50mL and are settled to 4L.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis storage liquid: 30% (W/V) acrylamide, 0.8% (W/V) N, N '-methylene diacrylamide;
The separation gel preparation of 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the H of 1.1mL
2Tutofusin tris-hydrogenchloride (pH:8.8) of 30% (V/V) sodium dodecyl sulfate-polyacrylamide gel electrophoresis storage liquid of O, 2.5mL, the 1.5mol/L of 1.3mL, 10% (W/V) sodium laurylsulfonate of 0.05mL, 10% (W/V) ammonium persulphate of 0.05mL, the N of 0.002mL, N, N ', N '-Tetramethyl Ethylene Diamine;
The separation gel preparation of 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the H of 0.68mL
2Tutofusin tris-hydrogenchloride (pH:6.8) of 30% (V/V) sodium dodecyl sulfate-polyacrylamide gel electrophoresis storage liquid of O, 0.17mL, the 0.5mol/L of 0.13mL, 10% (W/V) sodium laurylsulfonate of 0.01mL, 10% (W/V) ammonium persulphate of 0.01mL, the N of 0.001mL, N, N ', N '-Tetramethyl Ethylene Diamine;
Deposition condition: 100 volts of constant voltages, about 2 hours of electrophoresis time.
Xylene Brilliant Cyanine G destainer: 25% dehydrated alcohol, 8% glacial acetic acid.
Coomassie brilliant blue staining liquid: the Xylene Brilliant Cyanine G G-250 of 0.6 gram is dissolved in the destainer of 300mL, filters, brown bottle stores;
After electrophoresis finishes gel film is placed coomassie brilliant blue staining liquid 4h, till being dipped to background colour and fading fully with destainer again.
Embodiment 8
Determining of garbanzo bean cotyledon active protein that obtains by the method for the invention and polypeptide anti-tumor activity:
Get and cultivated 4-5 days, be in one bottle of the cell cultures of exponential phase of growth, add an amount of trypsinase-edta solution, attached cell is come off, the U.S.'s Sigma RPMI-1640 that contains 5% foetal calf serum with 10mL is made into suspension;
Do the cell counting with trypan blue dyeing back on blood counting chamber, general non-staining viable cell should be more than 97%;
With perfect medium diluting cells suspension, be made into every 100mL and contain 5000-40000 cell;
Get 96 hole flat boards, every hole adds cell suspension 100 μ L, and the process that adds cell should be controlled in 4 hours, and flat board is placed 37 ℃ of CO
2(5%) incubator is 24 hours;
Test-compound is made 5 extent of dilution, and peak concentration is 10-4mol/L, do successively dilution in 1: 10 (10-4,10-5,10-6,10-7,10-8mol/L).The crude extract peak concentration of natural product is also done dilution in 1: 10 with 250 μ g/mL.Sample does not generally need thermal sterilization or filtration, for fear of pollution, adds 50 μ g/mL gentamicins in the perfect medium that dilute sample is used;
Flat board is existed, contain 5%CO
2Hatched in the incubator of air and 100% humidity 2-4 days;
3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt is made into 1mg/mL solution with serum-free U.S. Sigma RPMI-1640, and every hole adds 50,37 ℃ of incubations 4 hours, makes MTT be reduced to the Jia Za;
The sucking-off supernatant liquor adds the dissolving of 150 μ L dimethyl sulfoxide (DMSO) Shi Jia Za, shakes up with dull and stereotyped shaking table;
With the dull and stereotyped reader of automatization spectrophotometric (Model312, BiotechResearchLaboratories, Inc., Rockville is Md) in the optical density(OD) of 550nm place each aperture of mensuration.
Interpretation of result
The survival rate of cell: the OD value of each test hole is deducted background OD value, and (perfect medium adds 3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt, acellular) or blank medicine hole OD value (perfect medium adds and is subjected to the different extent of dilution of reagent thing to add 3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt, acellular), the OD value of each repeating hole is got mean ± SD.
The survival rate of cell represents that with T/C% T is the OD value of dosing cell, and C is the OD value of control cells.
Cell survival rate %=(dosing cell OD/ control cells OD) * 100
Drug level (IC when obtaining T/C=50
50).
The IC of gained protein and polypeptide
50Concentration
Claims (5)
1. method of extracting natural antitumor protein and polypeptide from the garbanzo bean cotyledon is characterized in that following these steps to carrying out:
A, garbanzo cleaned remove foreign material, soak 1h-8h;
B, the garbanzo that step a is cleaned, soaks are put into the bean sprouts case and are germinateed, and temperature is 15-30 ℃, and water temperature 15-30 ℃, trickle time 3-10 second, trickle 2-6 hour at interval, germinating time 3-7 days;
Bud on c, the garbanzo bean sprouts that step b is germinateed and bean cotyledon separately and dry respectively;
D, the step c exsiccant garbanzo bean cotyledon that dries in the air is pulverized, cross the 10-60 mesh sieve after, carry out the cold soaking degreasing with normal hexane, the gained spent meal is standby;
E, with steps d spent meal liquid nitrogen grinding, stir to extract with phosphoric acid buffer again, the time is 1-5 hour, filters, and filtrate is removed precipitation with refrigerated centrifuge, it is standby to obtain supernatant liquor;
F, step e supernatant liquor is added anhydrous slufuric acid ammonia, fully place refrigerator after the stirring and dissolving and left standstill low-temperature centrifugation 24 hours, time 20min will precipitate with behind the water dissolution dialysis desalting, carry out low-temperature centrifugation again, time 20min, the precipitation drying can obtain anti-tumor active protein matter and polypeptide;
G, utilize the protein that the ordinary method sodium dodecyl sulfate-polyacrylamide gel electrophoresis determine to extract and the composition and the molecular weight thereof of polypeptide again, the activity that the protein that extracts and polypeptide suppress colon cancer cell is determined the protein of extraction and the anti-tumor activity of polypeptide.
2. method according to claim 1, it is characterized in that among step e, f and the g extraction, separate, purge process institute water is tri-distilled water.
3. method according to claim 1 and 2 is characterized in that the low-temperature centrifugation speed among step e, the f is 12000 rev/mins.
4. method according to claim 1 is characterized in that concentration of phosphate buffer is 0.01-0.1mol/L among the step e, pH=4.0-10.0.
5. method according to claim 1 is characterized in that anhydrous slufuric acid ammonium saturation ratio is 30%-80% among the step f.
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WO2006094450A1 (en) * | 2005-03-07 | 2006-09-14 | Jumpsun Bio-Medicine (Shanghai) Co., Ltd. | Medicaments and food for treatment or prevention of obesity and/or diabetes containing cicer arietinum extract |
CN101602799A (en) * | 2009-07-21 | 2009-12-16 | 中国科学院新疆理化技术研究所 | The fast preparation method of primary antimicrobial polypeptide in a kind of garbanzo |
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CN101602799A (en) * | 2009-07-21 | 2009-12-16 | 中国科学院新疆理化技术研究所 | The fast preparation method of primary antimicrobial polypeptide in a kind of garbanzo |
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《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 20080515 崔竹梅 鹰嘴豆蛋白的制备与其蛋白肽功能性质的研究 , * |
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CN104761617A (en) * | 2015-04-21 | 2015-07-08 | 中国科学院新疆理化技术研究所 | Preparation method and application of chickpea polypeptide part |
CN104761617B (en) * | 2015-04-21 | 2020-04-10 | 中国科学院新疆理化技术研究所 | Preparation method and application of chickpea polypeptide part |
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