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
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- protein
- garbanzo
- bean cotyledon
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 92
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 89
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 87
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 83
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 83
- 244000045195 Cicer arietinum Species 0.000 title claims abstract description 67
- 235000010523 Cicer arietinum Nutrition 0.000 title claims abstract description 65
- 235000010627 Phaseolus vulgaris Nutrition 0.000 title claims abstract description 61
- 244000046052 Phaseolus vulgaris Species 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 43
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 27
- 238000000605 extraction Methods 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 11
- 238000011033 desalting Methods 0.000 claims abstract description 9
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000005119 centrifugation Methods 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 17
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- 238000001556 precipitation Methods 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 16
- 238000005238 degreasing Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000002253 acid Substances 0.000 claims description 10
- 235000012054 meals Nutrition 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 229910021529 ammonia Inorganic materials 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000010926 purge Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 2
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 101000972324 Cynodon dactylon Leaf protein Proteins 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 3
- 231100000956 nontoxicity Toxicity 0.000 abstract description 3
- 239000000546 pharmaceutical excipient Substances 0.000 abstract description 3
- 239000008055 phosphate buffer solution Substances 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 2
- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 235000013376 functional food Nutrition 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 73
- 238000002360 preparation method Methods 0.000 description 24
- 239000003814 drug Substances 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 238000004140 cleaning Methods 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 7
- 239000000654 additive Substances 0.000 description 7
- 230000000996 additive effect Effects 0.000 description 7
- 238000010612 desalination reaction Methods 0.000 description 7
- 230000035784 germination Effects 0.000 description 6
- 239000000843 powder Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 3
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- PKZCRWFNSBIBEW-UHFFFAOYSA-N 2-n,2-n,2-trimethylpropane-1,2-diamine Chemical compound CN(C)C(C)(C)CN PKZCRWFNSBIBEW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 239000004160 Ammonium persulphate Substances 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 101001135767 Rattus norvegicus Parathyroid hormone Proteins 0.000 description 2
- 101710162629 Trypsin inhibitor Proteins 0.000 description 2
- 229940122618 Trypsin inhibitor Drugs 0.000 description 2
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 235000019395 ammonium persulphate Nutrition 0.000 description 2
- 229940088623 biologically active substance Drugs 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- AYRVGWHSXIMRAB-UHFFFAOYSA-M sodium acetate trihydrate Chemical compound O.O.O.[Na+].CC([O-])=O AYRVGWHSXIMRAB-UHFFFAOYSA-M 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000002753 trypsin inhibitor Substances 0.000 description 2
- QQGISFDJEJMKIL-JAIQZWGSSA-N (5z)-5-[[3-(hydroxymethyl)thiophen-2-yl]methylidene]-10-methoxy-2,2,4-trimethyl-1h-chromeno[3,4-f]quinolin-9-ol Chemical class C1=CC=2NC(C)(C)C=C(C)C=2C2=C1C=1C(OC)=C(O)C=CC=1O\C2=C/C=1SC=CC=1CO QQGISFDJEJMKIL-JAIQZWGSSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 206010061623 Adverse drug reaction Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 235000010521 Cicer Nutrition 0.000 description 1
- 241000220455 Cicer Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 206010024419 Libido decreased Diseases 0.000 description 1
- 241001300479 Macroptilium Species 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 241000594182 Sarcophaga sigma Species 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108010025899 gelatin film Proteins 0.000 description 1
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical class O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000002221 olecranon process Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 235000021251 pulses Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000008261 skin carcinoma Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940108519 trasylol Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Plant Substances (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105028096A CN101962399B (en) | 2010-10-11 | 2010-10-11 | Method for extracting natural antitumor active protein and polypeptide from chickpea bean |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105028096A CN101962399B (en) | 2010-10-11 | 2010-10-11 | Method for extracting natural antitumor active protein and polypeptide from chickpea bean |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101962399A true CN101962399A (en) | 2011-02-02 |
| CN101962399B CN101962399B (en) | 2012-07-11 |
Family
ID=43515461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105028096A Expired - Fee Related CN101962399B (en) | 2010-10-11 | 2010-10-11 | Method for extracting natural antitumor active protein and polypeptide from chickpea bean |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101962399B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2010
- 2010-10-11 CN CN2010105028096A patent/CN101962399B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 《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 , * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101962399B (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109303922B (en) | Rosa roxburghii polysaccharide functionalized nano-selenium compound, preparation method thereof and application thereof in hypoglycemic drugs | |
| CN106831375B (en) | Chalcone compound with anti-tumor activity and preparation method and application thereof | |
| CN107309438B (en) | Method for preparing nano-silver composite particles from phyllanthus emblica polysaccharide | |
| CN107988301B (en) | Preparation method and application of chickpea bean cotyledon polypeptide | |
| CN108752466A (en) | A kind of chelated calcium preparation method of tuna bone collagen peptide | |
| CN104667289B (en) | A kind of antineoplastic drug carrier and its application method | |
| CN103289948B (en) | The application of a kind of GF microcarrier for cell culture in anchorage-dependent cells is cultivated | |
| WO2016041258A1 (en) | Method for preparing bamboo fungus polysaccharide-zinc chelate and use thereof | |
| CN103275923B (en) | A kind of GF microcarrier for cell culture and preparation method thereof | |
| CN110934922A (en) | Total flavone in lycium ruthenicum, extraction method and application | |
| CN109232760A (en) | Phellinus protect liver polysaccharide PPB-2 and preparation method thereof | |
| CN110642955A (en) | Esterified selenium polysaccharide and preparation method and application thereof | |
| CN106075384B (en) | Pea active peptide is inhibiting the application and preparation method thereof in growth of cancer cells | |
| CN101962399B (en) | Method for extracting natural antitumor active protein and polypeptide from chickpea bean | |
| CN102698282A (en) | Preparation method for multi-responsive magnetic composite nanoparticles for loading medicament | |
| CN101670116B (en) | Forebody drug with conjugate linoleic acid connected with antitumor drug and preparation method thereof | |
| CN103861104A (en) | Phycocyanin-phthalocyanine compound and preparation method and application thereof | |
| CN112481341A (en) | Preparation method and application of selenium absorption enhancing active peptide | |
| CN107868019A (en) | Dihydro oat alkaloid compound, derivative and its preparation method and application | |
| CN102234258A (en) | Preparation method and application of sphaelactone | |
| CN104096231A (en) | Targeting nano sono-sensitizer and preparation method thereof | |
| CN109022309B (en) | Stenotrophomonas maltophilia capable of producing free fatty acid and application thereof | |
| CN102000132B (en) | Preparation method of traditional Chinese medicine preparation and antioxidation application thereof | |
| CN104211815B (en) | A kind of ferritin heavy chain subunit nano medicament carrying system and preparation method and application | |
| CN108514546B (en) | Chitosan hydrogel preparation of dihydromyricetin and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120711 |