CN107574209B - 一种高精氨酸混合肽的制备方法及其在***治疗中的应用 - Google Patents
一种高精氨酸混合肽的制备方法及其在***治疗中的应用 Download PDFInfo
- Publication number
- CN107574209B CN107574209B CN201610520561.3A CN201610520561A CN107574209B CN 107574209 B CN107574209 B CN 107574209B CN 201610520561 A CN201610520561 A CN 201610520561A CN 107574209 B CN107574209 B CN 107574209B
- Authority
- CN
- China
- Prior art keywords
- mixed
- homoarginine
- peptide
- mixed peptide
- enzymolysis
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 124
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 title claims abstract description 61
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 206010008342 Cervix carcinoma Diseases 0.000 title claims abstract description 15
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 title claims abstract description 15
- 201000010881 cervical cancer Diseases 0.000 title claims abstract description 15
- 239000012528 membrane Substances 0.000 claims abstract description 47
- 239000004475 Arginine Substances 0.000 claims abstract description 27
- 239000000725 suspension Substances 0.000 claims abstract description 24
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 21
- YIKSHDNOAYSSPX-UHFFFAOYSA-N 1-propan-2-ylthioxanthen-9-one Chemical compound S1C2=CC=CC=C2C(=O)C2=C1C=CC=C2C(C)C YIKSHDNOAYSSPX-UHFFFAOYSA-N 0.000 claims abstract description 13
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims abstract description 13
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims abstract description 13
- HWSSEYVMGDIFMH-UHFFFAOYSA-N 2-[2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOCCOC(=O)C(C)=C HWSSEYVMGDIFMH-UHFFFAOYSA-N 0.000 claims abstract description 13
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000020234 walnut Nutrition 0.000 claims abstract description 13
- 235000009496 Juglans regia Nutrition 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 10
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 10
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 10
- 239000000178 monomer Substances 0.000 claims abstract description 9
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 8
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 7
- 108090000526 Papain Proteins 0.000 claims abstract description 7
- 239000004365 Protease Substances 0.000 claims abstract description 7
- 229940055729 papain Drugs 0.000 claims abstract description 7
- 235000019834 papain Nutrition 0.000 claims abstract description 7
- 239000011521 glass Substances 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000000843 powder Substances 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- 239000011259 mixed solution Substances 0.000 claims description 18
- 239000006059 cover glass Substances 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 12
- 241000758789 Juglans Species 0.000 claims description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 229940088598 enzyme Drugs 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 238000003825 pressing Methods 0.000 claims description 8
- 238000001694 spray drying Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 235000012054 meals Nutrition 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000002444 silanisation Methods 0.000 claims description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 5
- 241000252506 Characiformes Species 0.000 claims description 5
- 150000001450 anions Chemical class 0.000 claims description 5
- 239000003729 cation exchange resin Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000012466 permeate Substances 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 3
- 230000009849 deactivation Effects 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- JOYLKPVNHPKSQC-UHFFFAOYSA-N methanol;3-triethoxysilylpropan-1-amine Chemical compound OC.CCO[Si](OCC)(OCC)CCCN JOYLKPVNHPKSQC-UHFFFAOYSA-N 0.000 claims 1
- 239000012982 microporous membrane Substances 0.000 claims 1
- 238000005070 sampling Methods 0.000 claims 1
- 230000035755 proliferation Effects 0.000 abstract description 8
- 208000019065 cervical carcinoma Diseases 0.000 abstract description 6
- 230000009471 action Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract description 2
- 240000007049 Juglans regia Species 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000003999 initiator Substances 0.000 abstract 1
- 230000000977 initiatory effect Effects 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 16
- 210000004881 tumor cell Anatomy 0.000 description 14
- 201000011510 cancer Diseases 0.000 description 13
- 229940024606 amino acid Drugs 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 8
- 235000013305 food Nutrition 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000001276 controlling effect Effects 0.000 description 5
- 102000007079 Peptide Fragments Human genes 0.000 description 4
- 108010033276 Peptide Fragments Proteins 0.000 description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 230000007480 spreading Effects 0.000 description 4
- 238000003892 spreading Methods 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 238000007605 air drying Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000009702 cancer cell proliferation Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 229920000768 polyamine Polymers 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 241000758791 Juglandaceae Species 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 1
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000000089 atomic force micrograph Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 235000020627 health maintaining nutrition Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/175—Amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/52—Juglandaceae (Walnut family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/362—Cation-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3852—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using imprinted phases or molecular recognition; using imprinted phases
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/13—Preparation or pretreatment of starting material involving cleaning, e.g. washing or peeling
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Water Supply & Treatment (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种高精氨酸含量的混合肽及其在***治疗中的应用。所述制备方法包括如下步骤:利用超高压对核桃粕和蛋清蛋白混悬液进行预处理,其后在超声波、微波辅助作用下使用碱性蛋白酶、木瓜蛋白酶分步酶解预处理产物;酶解液过滤后的清液经反相高效液相色谱法分离其中的目标混合肽;以此混合肽为模板分子,丙烯酸、甲基丙烯酸为功能单体,三乙二醇二甲基丙烯酸酯为交联剂,异丙基硫杂蒽酮为引发剂,紫外引发聚合形成表面分子印迹膜,用于分离富集酶解清液中的目标混合肽。所述混合肽中精氨酸含量在18%以上;经MTT法检测发现,所述高精氨酸混合肽能有效抑制人***Hela细胞的增殖。本法有助于降低产品成本,加速实现工业化生产。
Description
技术领域
本发明涉及一种高精氨酸混合肽的制备方法及其在***治疗中的应用,具体涉及一种以核桃粕、蛋清蛋白为原料制备富含精氨酸的混合肽的方法及其在***治疗中的应用。
背景技术
癌症是恶性肿瘤的统称,特点是机体细胞失去正常的调节控制,过度增殖,同时有不同程度的分化障碍,并常侵犯邻近组织或转移到远离部位。它严重威胁全人类健康,位居非正常死亡原因之首。世界卫生组织(WHO)2014年发布的《全球癌症报告》显示,2012年中国的新增癌症病例高居全球第一位。2015年,国家卫计委、国家***等16个部门联合发布的《中国癌症防治三年行动计划(2015-2017年)》中明确指出,我国的癌症防治要以最高发、危害最大的8项癌症为重点,而***即是我国八大癌之一,为女性常发癌之一。
针对恶性肿瘤,传统的治疗方法包括手术切除、化疗、放疗等。这些方法在一定程度上起到了***的作用,但一旦发生癌细胞局部扩散或潜在转移,治疗往往以失败告终。且这些传统疗法不可避免地会对人体正常细胞、自身免疫***造成不同程度的伤害,引起呕吐、食欲不振、头痛失眠、脱发溃疡、糜烂发炎、血象下降等不良反应,病人常因身体难以承受而无法继续接受治疗,严重影响癌症患者生存质量。
现代科学研究发现,许多天然生物活性肽对抑制肿瘤细胞增殖和转移具有良好功效,且选择性高、毒副作用小。这些活性肽通过诱导肿瘤分化、抑制肿瘤细胞生长、抑制肿瘤新生血管生成、提高肿瘤细胞对药物的敏感性、降低化疗损伤、增强药物对肿瘤细胞的杀伤力、干扰肿瘤细胞DNA合成、提高机体免疫力等作用达到治疗或辅助治疗恶性肿瘤的目的。近年来不断有研究证实,增量精氨酸对肿瘤细胞的抑制及诱导凋亡作用显著。肿瘤细胞中多胺代谢活跃,多胺是其迅速***、增殖必需物质,增量精氨酸能通过抑制鸟氨酸脱羧酶活性来抑制多胺的生物合成,从而抑制癌细胞增殖;精氨酸经一氧化氮合酶作用后生成NO,NO可阻断肿瘤细胞的能量代谢和DNA复制,或者引起肿瘤细胞DNA损伤,从而抑制肿瘤细胞的生长或引起肿瘤细胞死亡;同时,NO可抑制细胞黏附分子的表达,从而阻止细胞的黏附,在肿瘤细胞脱离原发组织,转移至其它部位形成新病灶的过程中,这些黏附分子起着重要作用;此外,精氨酸还能增加T淋巴细胞的产生和功能,增加体内白介素IL-2的生成及其受体表达,调节和激活体内巨噬细胞活性,从而提高肿瘤宿主自身免疫力。国外已有研究开始尝试对癌症患者采用不平衡氨基酸疗法,通过调整肿瘤宿主体内某些氨基酸的含量,干扰肿瘤细胞的代谢和功能,进而抑制肿瘤的生长或诱导其凋亡。另有报道称,富含精氨酸等亲水性氨基酸的亲水性多肽,可通过静电吸引方式,特异性作用于肿瘤细胞,导致细胞膜迅速破裂,细胞内容物渗漏,最终引起细胞死亡;也有研究称多肽分子结构中的碱性氨基酸(精氨酸、赖氨酸等)对其抗肿瘤活性有一定影响。这些都为本发明所述的高精氨酸混合肽的抑癌功效提供了坚实的理论基础。
核桃粕是核桃加工副产品,蛋白质消化率高、氨基酸含量丰富且种类齐全,但在我国主要用于饲料工业,深度加工不够而造成资源贬值;鸡蛋清蛋白不仅富含人体8种必需氨基酸,且组成与人体蛋白的氨基酸组成接近,人对其消化利用率极高。本发明首次使用来自鸡蛋和核桃的动、植物混合蛋白为原料,通过超高压-超声-微波辅助酶解、表面分子印迹膜分离纯化等手段,制备富含精氨酸的活性混合肽,并将其应用于***治疗,在对患者进行营养补充的同时抑制癌细胞增殖,有效改善***患者的生存质量。与之相关的应用研究至今未见报道。
发明内容
本发明要解决的技术问题是提供一种新颖的高精氨酸混合肽的制备方法及其在***治疗中的应用。该混合肽富含精氨酸,具有较强的抑制***细胞增殖的作用。
为了解决上述技术问题,本发明提供了如下技术方案:
一种高精氨酸混合肽的制备方法,所述制备方法包括如下步骤:
(1):将经脱脂、粉碎的核桃粕、鸡蛋清蛋白与水混匀,搅拌后超高压预处理,随后超声波-微波辅助酶解,酶解结束后升温灭酶,板框压滤收集清液;
(2):清液冻干后,利用反相高效液相色谱法(RP-HPLC)对冻干粗粉中的目标肽段进行分离。采用Everest C18(4.6×250mm,5μm,238EV54)作为反相柱,乙腈水溶液作为流动相,三氟乙酸作为阴离子对试剂,检测波长214nm,上样前纯乙腈冲洗色谱柱,流动相溶解25mg冻干粉定容至25mL,0.45μm微孔滤膜过滤,进样体积20μL,柱温30℃,分离条件为乙腈浓度18%(v/v),三氟乙酸浓度0.09%(v/v),流速1.0mL/min,保留时间为9.64min、11.36min、13.80min,收集三个洗脱组分,冻干后即为高精氨酸混合肽粉;
优选地,所述高精氨酸混合肽的制备方法还包括表面印迹膜分离富集高精氨酸混合肽的步骤,所述步骤为:
(3):用piranha溶液(体积比3:1的浓硫酸和30%双氧水)浸没盖玻片和载玻片,超声清洗1.5~2.5h后,纯水洗净,氮气吹干待用;将洗净的盖玻片浸泡于高精氨酸混合肽水溶液中以获得混合肽固定化模板;将洗净的载玻片浸泡于0.5~1.5%(v/v)3-氨丙基三乙氧基硅烷(APTES)的甲醇溶液中,20~40rpm振荡15~45min后,甲醇漂洗晾干,获得硅烷化载玻片;将功能单体丙烯酸(AA)、甲基丙烯酸(MAA),交联剂三乙二醇二甲基丙烯酸酯(TEGDMA)混匀,加入光引发剂异丙基硫杂蒽酮(ITX)获得预聚混合液;向预聚混合液中通入氮气后将其平铺于硅烷化载玻片表面,载玻片经旋转后将混合肽固定化模板覆盖于其上,紫外光引发聚合完成后,将玻片浸泡于8~12%(m/v)SDS:8~12%(v/v)HAc溶液中,揭去盖玻片,80~160rpm振荡4~8h后,纯水振荡漂洗至中性,获得高精氨酸混合肽表面印迹膜;
(4):将步骤(3)获得的高精氨酸混合肽表面印迹膜浸没于步骤(1)获得的清液中,20~40rpm振荡1~6h后取出吸附了目标肽的印迹膜,浸没于0.5~1.6mol/L NaCl溶液中,同时100~300W超声10~50min辅助洗脱;收集洗脱液,通过阳离子交换树脂去除NaCl,透过液经低温喷雾干燥获得高精氨酸混合肽粉末;洗脱后的表面印迹膜经纯水漂洗,再次浸没于步骤(1)获得的新的清液中,重复后续步骤,用于分离高精氨酸混合肽。
优选地,步骤(1)中,将经脱脂粉碎的核桃粕与鸡蛋清蛋白按质量比3~6:1混合,将此蛋白粕混合物与水按质量体积比1:4~14混匀,室温搅拌1.5~2.5h后置于超高压主机***中,处理压力200~600MPa,保压时间10~30min,获得经超高压预处理的混悬液;将混悬液控温至40~60℃,调pH至9~10,加入混悬液质量2%~6%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200~400W,超声时间10~20min,微波功率200~600W,微波时间5~15min,酶解1.5~2.5h后调pH至6~8,加入混悬液质量2%~6%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200~400W,超声时间10~20min,微波功率200~600W,微波时间5~15min,酶解2~3h后升温灭酶,板框压滤后收集清液。
优选地,步骤(3)中,将洗净的盖玻片浸泡于1.5~10g/L高精氨酸混合肽水溶液中,20~40rpm振荡4~8h后,纯水漂洗晾干,获得混合肽固定化模板。
优选地,步骤(3)中,将功能单体丙烯酸(AA)、甲基丙烯酸(MAA),交联剂三乙二醇二甲基丙烯酸酯(TEGDMA)按体积比0.5~3.5:0.5~2.5:4~11混匀,加入0.2~0.8体积1~4mmol/L的异丙基硫杂蒽酮(ITX)的丙酮溶液作为光引发剂,获得预聚混合液。
优选地,步骤(3)中,向预聚混合液中通入氮气20~40min后,将其平铺在固定于旋转机上的硅烷化载玻片表面,旋转机100~400rpm旋转2~10s后,将混合肽固定化模板覆盖于载玻片上,365nm紫外光引发聚合3~6h。
优选地,步骤(4)中,高精氨酸混合肽表面印迹膜重复吸附-洗脱过程10次以上。
本发明还提供一种高精氨酸混合肽,所述高精氨酸混合肽通过上述制备方法制备。
优选地,所述混合肽中精氨酸含量在18%以上。
本发明还提供经上述制备方法得到的高精氨酸混合肽在制备与***治疗相关的保健食品、特殊膳食用食品、普通食品及药品中的应用。
本发明的有益效果如下:
从功效角度考虑,本发明制备的高精氨酸混合肽,能明显抑制人***细胞的增殖,藉由控制肿瘤的生长来延缓***的恶化;癌症患者面临的一个主要问题是营养障碍,本发明制备的混合肽,有助于减轻肠道消化过程中游离氨基酸相互竞争共同吸收位点而产生的吸收抑制,大量科学研究已证实,当以肽形式作为氮源时,整体蛋白质沉积高于相应的游离氨基酸摄入或完整蛋白质摄入。本发明所述高精氨酸混合肽,在对患者进行营养补充的同时抑制癌细胞增殖,可有效改善***患者的生存质量。
从安全角度考虑,在《GB29922-2013食品安全国家标准特殊医学用途配方食品通则》中规定,不得使用非食用的动植物水解原料作为单体氨基酸的来源,本发明制备的高精氨酸混合肽来源于核桃、鸡蛋等天然食材,符合现今推崇的天然健康的营养理念;而水解用酶亦符合《GB 2760-2014食品安全国家标准食品添加剂使用标准》中的相关规定。
从制备角度考虑,本发明的制备方法条件温和,涉及仪器均为食药行业常用设备;表面印迹膜吸附-洗脱效果佳,重复利用率高;所述制备方法有利于降低生产成本,极具扩大化生产潜力,有助于加速实现产品的工业化量产。
附图说明
图1为本发明高精氨酸混合肽制备流程图;
图2为本发明高精氨酸混合肽高效液相色谱图;
图3为本发明高精氨酸混合肽表面印迹膜制备示意图;
图4为本发明高精氨酸混合肽固定化模板原子力显微镜图;
图5为本发明高精氨酸混合肽表面印迹膜扫描电镜图;
图6为本发明高精氨酸混合肽表面印迹膜重复利用性。
具体实施方式
以下通过具体的实施例进一步说明本发明,但以下实施例仅用于说明本发明而非用于限制本发明。
实施例1:
本发明所述高精氨酸混合肽的制备方法,包括以下步骤:
1、将经脱脂、粉碎的核桃粕与鸡蛋清蛋白按质量比4:1混合,将此蛋白粕混合物与水按质量体积比1:8混匀,室温搅拌2h后置于超高压主机***中,处理压力400MPa,保压时间20min,获得经超高压预处理的混悬液;将混悬液控温至50℃,调pH至9,加入混悬液质量3.5%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率300W,超声时间12min,微波功率400W,微波时间8min,酶解2h后调pH至7,加入混悬液质量3.5%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率300W,超声时间15min,微波功率400W,微波时间10min,酶解2.5h后升温灭酶,板框压滤后收集清液。其蛋白质含量为93.6%,肽含量为88.5%;
2、清液冻干后,利用反相高效液相色谱法(RP-HPLC)对冻干粗粉中的目标肽段进行分离。采用Everest C18(4.6×250mm,5μm,238EV54)作为反相柱,乙腈水溶液作为流动相,三氟乙酸作为阴离子对试剂,检测波长214nm,上样前纯乙腈冲洗色谱柱,流动相溶解25mg冻干粉定容至25mL,0.45μm微孔滤膜过滤,进样体积20μL,柱温30℃,分离条件为乙腈浓度18%(v/v),三氟乙酸浓度0.09%(v/v),流速1.0mL/min,保留时间为9.64min、11.36min、13.80min,收集三个洗脱组分,冻干后即为高精氨酸混合肽粉,目标肽含量占粗粉23.6%;
3、用piranha溶液(体积比3:1的浓硫酸和30%双氧水)浸没玻片(盖玻片和载玻片),超声清洗2h后,纯水洗净,氮气吹干待用;将洗净的盖玻片浸泡于5g/L高精氨酸混合肽水溶液中,20rpm振荡6h后,纯水漂洗晾干,获得混合肽固定化模板;将洗净的载玻片浸泡于1%(v/v)3-氨丙基三乙氧基硅烷(APTES)的甲醇溶液中,20rpm振荡30min后,甲醇漂洗晾干,获得硅烷化载玻片;将功能单体丙烯酸(AA)、甲基丙烯酸(MAA),交联剂三乙二醇二甲基丙烯酸酯(TEGDMA)按体积比2:1:7混匀,加入0.5体积2.5mmol/L的异丙基硫杂蒽酮(ITX)的丙酮溶液作为光引发剂,获得预聚混合液;向预聚混合液中通入氮气30min后,将其平铺在固定于旋转机上的硅烷化载玻片表面,旋转机200rpm旋转4s后,将混合肽固定化模板覆盖于载玻片上,365nm紫外光引发聚合4h;将聚合完成后的玻片浸泡于10%(m/v)SDS:10%(v/v)HAc溶液中,揭去盖玻片,120rpm振荡6h后,纯水振荡漂洗至中性,获得高精氨酸混合肽表面印迹膜;
4、将步骤3获得的高精氨酸混合肽表面印迹膜浸没于步骤1获得的清液中,20rpm振荡4h后取出吸附了目标肽的印迹膜,浸没于1mol/L NaCl溶液中,同时200W超声30min辅助洗脱,洗脱液通过阳离子交换树脂去除NaCl,透过液经低温喷雾干燥获得高精氨酸混合肽粉末。表面印迹膜吸附率为80.5%(见表1),喷雾干燥获得的混合肽中精氨酸含量为21.3%(见表2);洗脱后的表面印迹膜经纯水漂洗,再次浸没于步骤1获得的新的清液中,重复后续步骤,用于分离高精氨酸混合肽,20次重复利用,再生率为91.1%(见图6);
5、采用高效液相色谱法测定表面印迹膜对板框压滤后清液中目标肽的吸附率,平行测定三次,计算公式为:吸附率(%)=洗脱液中目标肽含量/清液中目标肽含量×100%,结果见表1:
表1高精氨酸混合肽表面印迹膜吸附率
高精氨酸混合肽表面印迹膜经吸附-洗脱-再吸附过程,重复利用20次后,其吸附量由最初的7.38mg/g降至6.72mg/g,再生率达91.1%,显示出极佳的重复利用性。
6、采用氨基酸分析仪对高精氨酸混合肽酸解后各氨基酸含量进行检测,结果见表2:
表2高精氨酸混合肽氨基酸分析结果
注:酸解后色氨酸、天冬酰胺、谷氨酰胺被破坏。
实施例2:
本发明所述混合肽的制备方法,包括以下步骤:
1、将经脱脂、粉碎的核桃粕与鸡蛋清蛋白按质量比3:1混合,将此蛋白粕混合物与水按质量体积比1:6混匀,室温搅拌1.5h后置于超高压主机***中,处理压力300MPa,保压时间15min,获得经超高压预处理的混悬液;将混悬液控温至50℃,调pH至9,加入混悬液质量3%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200W,超声时间15min,微波功率300W,微波时间10min,酶解1.5h后调pH至7,加入混悬液质量3%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200W,超声时间18min,微波功率300W,微波时间12min,酶解2h后升温灭酶,板框压滤后收集清液。其蛋白质含量为90.3%,肽含量为81.7%;
2、清液冻干后,利用反相高效液相色谱法(RP-HPLC)对冻干粗粉中的目标肽段进行分离。采用Everest C18(4.6×250mm,5μm,238EV54)作为反相柱,乙腈水溶液作为流动相,三氟乙酸作为阴离子对试剂,检测波长214nm,上样前纯乙腈冲洗色谱柱,流动相溶解25mg冻干粉定容至25mL,0.45μm微孔滤膜过滤,进样体积20μL,柱温30℃,分离条件为乙腈浓度18%(v/v),三氟乙酸浓度0.09%(v/v),流速1.0mL/min,保留时间为9.64min、11.36min、13.80min,收集三个洗脱组分,冻干后即为高精氨酸混合肽粉,目标肽含量占粗粉19.8%;
3、用piranha溶液(体积比3:1的浓硫酸和30%双氧水)浸没玻片(盖玻片和载玻片),超声清洗1.5h后,纯水洗净,氮气吹干待用;将洗净的盖玻片浸泡于3g/L高精氨酸混合肽水溶液中,30rpm振荡4h后,纯水漂洗晾干,获得混合肽固定化模板;将洗净的载玻片浸泡于0.8%(v/v)3-氨丙基三乙氧基硅烷(APTES)的甲醇溶液中,30rpm振荡18min后,甲醇漂洗晾干,获得硅烷化载玻片;将功能单体丙烯酸(AA)、甲基丙烯酸(MAA),交联剂三乙二醇二甲基丙烯酸酯(TEGDMA)按体积比1.5:0.8:7混匀,加入0.3体积3mmol/L的异丙基硫杂蒽酮(ITX)的丙酮溶液作为光引发剂,获得预聚混合液;向预聚混合液中通入氮气20min后,将其平铺在固定于旋转机上的硅烷化载玻片表面,旋转机100rpm旋转6s后,将混合肽固定化模板覆盖于载玻片上,365nm紫外光引发聚合5h;将聚合完成后的玻片浸泡于8%(m/v)SDS:8%(v/v)HAc溶液中,揭去盖玻片,160rpm振荡4h后,纯水振荡漂洗至中性,获得高精氨酸混合肽表面印迹膜;
4、将步骤3获得的高精氨酸混合肽表面印迹膜浸没于步骤1获得的清液中,30rpm振荡5h后取出吸附了目标肽的印迹膜,浸没于0.8mol/L NaCl溶液中,同时100W超声40min辅助洗脱,洗脱液通过阳离子交换树脂去除NaCl,透过液经低温喷雾干燥获得高精氨酸混合肽粉末。表面印迹膜吸附率为71.3%,20次重复利用再生率为84.2%,喷雾干燥获得的混合肽中精氨酸含量为18.9%。
实施例3:
本发明所述混合肽的制备方法,包括以下步骤:
1、将经脱脂、粉碎的核桃粕颗粒与鸡蛋清蛋白按质量比5:1混合,将此蛋白粕混合物与水按质量体积比1:10混匀,室温搅拌2.5h后置于超高压主机***中,处理压力500MPa,保压时间25min,获得经超高压预处理的混悬液;将混悬液控温至50℃,调pH至9,加入混悬液质量4%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率400W,超声时间20min,微波功率500W,微波时间5min,酶解2.5h后调pH至7,加入混悬液质量4%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率400W,超声时间20min,微波功率500W,微波时间8min,酶解3h后升温灭酶,板框压滤后收集清液。其蛋白质含量为92.1%,肽含量为84.7%;
2、清液冻干后,利用反相高效液相色谱法(RP-HPLC)对冻干粗粉中的目标肽段进行分离。采用Everest C18(4.6×250mm,5μm,238EV54)作为反相柱,乙腈水溶液作为流动相,三氟乙酸作为阴离子对试剂,检测波长214nm,上样前纯乙腈冲洗色谱柱,流动相溶解25mg冻干粉定容至25mL,0.45μm微孔滤膜过滤,进样体积20μL,柱温30℃,分离条件为乙腈浓度18%(v/v),三氟乙酸浓度0.09%(v/v),流速1.0mL/min,保留时间为9.64min、11.36min、13.80min,收集三个洗脱组分,冻干后即为高精氨酸混合肽粉,目标肽含量占粗粉21.9%;
3、用piranha溶液(体积比3:1的浓硫酸和30%双氧水)浸没玻片(盖玻片和载玻片),超声清洗2.5h后,纯水洗净,氮气吹干待用;将洗净的盖玻片浸泡于4g/L高精氨酸混合肽水溶液中,40rpm振荡8h后,纯水漂洗晾干,获得混合肽固定化模板;将洗净的载玻片浸泡于1.2%(v/v)3-氨丙基三乙氧基硅烷(APTES)的甲醇溶液中,40rpm振荡40min后,甲醇漂洗晾干,获得硅烷化载玻片;将功能单体丙烯酸(AA)、甲基丙烯酸(MAA),交联剂三乙二醇二甲基丙烯酸酯(TEGDMA)按体积比3:1.5:8混匀,加入0.6体积4mmol/L的异丙基硫杂蒽酮(ITX)的丙酮溶液作为光引发剂,获得预聚混合液;向预聚混合液中通入氮气40min后,将其平铺在固定于旋转机上的硅烷化载玻片表面,旋转机300rpm旋转3s后,将混合肽固定化模板覆盖于载玻片上,365nm紫外光引发聚合6h;将聚合完成后的玻片浸泡于10%(m/v)SDS:8%(v/v)HAc溶液中,揭去盖玻片,140rpm振荡8h后,纯水振荡漂洗至中性,获得高精氨酸混合肽表面印迹膜;
4、将步骤3获得的高精氨酸混合肽表面印迹膜浸没于步骤1获得的清液中,40rpm振荡6h后取出吸附了目标肽的印迹膜,浸没于1.2mol/L NaCl溶液中,同时300W超声20min辅助洗脱,洗脱液通过阳离子交换树脂去除NaCl,透过液经低温喷雾干燥获得高精氨酸混合肽粉末。表面印迹膜吸附率为77.5%,20次重复利用再生率为88.0%,喷雾干燥获得的混合肽中精氨酸含量为20.4%。
实施例4:采用MTT法评价高精氨酸混合肽对人***细胞增殖的抑制作用
1、仪器与材料
METERTECH∑960酶标仪为台湾METERTECH公司产品;(MCO-15AC)CO2细胞培养箱为日本三洋公司公司产品;1300SERIES A2生物安全柜为美国Thermo Fisher公司产品;TDL-50B低速台式离心机为上海安亭科学仪器厂产品;D-1型自动蒸汽灭菌锅为北京发恩科贸有限公司产品。
人***Hela细胞购自中国科学院细胞库;RPMI-1640培养基购自美国GIBCO公司;胎牛血清购自天津市川页生化制品有限公司;细胞增殖及细胞毒性检测试剂盒(MTT)购自上海翊圣生物科技有限公司;高精氨酸混合肽为实施例1制备所得。
2、实验方法
取对数生长期的人***Hela细胞,配成3×104个/mL浓度的单细胞悬液接种于96孔板中,100μL/孔,置于37℃、5%CO2培养箱中培养24h贴壁后弃培养液。设对照组和实验组,实验组分别加入实施例1制备得到的不同浓度的高精氨酸混合肽(4mg/mL、6mg/mL和8mg/mL,用RPMI-1640培养基稀释),对照组加入等体积RPMI-1640培养基。培养24h后加入MTT工作液(5mg/mL、10μL/孔),4h后沿培养液上部吸去100μL上清液,加入100μL甲臜溶解液,继续培养4h后用酶标仪测定吸光度(波长570nm),计算高精氨酸混合肽对细胞增殖的抑制率,每组设4个重复孔。
抑制率(%)=(对照组A570—实验组A570)/对照组A570×100%
3、实验结果
由表3可知,3个不同浓度(4mg/mL、6mg/mL和8mg/mL)的高精氨酸混合肽作用Hela细胞后,对Hela细胞的增殖均具有明显的抑制,并且随着混合肽浓度增加,抑制率也随之增加。
4、实验结论
本发明高精氨酸混合肽具有较强的抑制***细胞增殖的作用,且在一定浓度范围内呈剂量依赖性。
应该理解,尽管参考其示例性的实施方案,已经对本发明进行具体地显示和描述,但是本领域的普通技术人员应该理解,在不背离由权利要求所定义的本发明的精神和范围的条件下,可以在其中进行各种形式和细节的变化,可以进行各种实施方案的任意组合。
Claims (9)
1.一种高精氨酸混合肽的制备方法,其特征在于,所述制备方法包括如下步骤:
(1):将经脱脂粉碎的核桃粕、鸡蛋清蛋白与水混匀,搅拌后超高压预处理,随后超声波-微波辅助酶解,酶解结束后升温灭酶,板框压滤收集清液;其中所述超声波-微波辅助酶解包括:在经超高压预处理的混悬液加入混悬液质量2%~6%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,酶解后经调pH至6-8,并加入混悬液质量2%~6%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解;
(2):清液冻干后,利用反相高效液相色谱法对冻干粗粉中的目标肽段进行分离,采用4.6×250mm,5μm,238EV54的Everest C18作为反相柱,乙腈水溶液作为流动相,三氟乙酸作为阴离子对试剂,检测波长214nm,上样前纯乙腈冲洗色谱柱,流动相溶解25mg冻干粉定容至25mL,0.45μm微孔滤膜过滤,进样体积20μL,柱温30℃,分离条件为乙腈浓度18%v/v,三氟乙酸浓度0.09%v/v,流速1.0mL/min,保留时间为9.64min、11.36min、13.80min,收集三个洗脱组分,冻干后即为高精氨酸混合肽粉;
(3):用体积比3:1的浓硫酸和30%双氧水的piranha溶液浸没盖玻片和载玻片,超声清洗1.5~2.5h后,纯水洗净,氮气吹干待用;将洗净的盖玻片浸泡于高精氨酸混合肽水溶液中以获得混合肽固定化模板;将洗净的载玻片浸泡于0.5~1.5%v/v 3-氨丙基三乙氧基硅烷的甲醇溶液中,20~40rpm振荡15~45min后,甲醇漂洗晾干,获得硅烷化载玻片;将功能单体丙烯酸、甲基丙烯酸,交联剂三乙二醇二甲基丙烯酸酯混匀,加入光引发剂异丙基硫杂蒽酮获得预聚混合液;向预聚混合液中通入氮气后将其平铺于硅烷化载玻片表面,载玻片经旋转后将混合肽固定化模板覆盖于其上,紫外光引发聚合完成后,将玻片浸泡于8~12%m/v SDS:8~12%v/v HAc溶液中,揭去盖玻片,80~160rpm振荡4~8h后,纯水振荡漂洗至中性,获得高精氨酸混合肽表面印迹膜;
(4):将步骤(3)获得的高精氨酸混合肽表面印迹膜浸没于步骤(1)获得的清液中,20~40rpm振荡1~6h后取出吸附了目标肽的印迹膜,浸没于0.5~1.6mol/L NaCl溶液中,同时100~300W超声10~50min辅助洗脱;收集洗脱液,通过阳离子交换树脂去除NaCl,透过液经低温喷雾干燥获得高精氨酸混合肽粉末;洗脱后的表面印迹膜经纯水漂洗,再次浸没于步骤(1)获得的新的清液中,重复后续步骤,用于分离高精氨酸混合肽。
2.根据权利要求1所述的高精氨酸混合肽的制备方法,其特征在于,步骤(1)中,将经脱脂粉碎的核桃粕与鸡蛋清蛋白按质量比3~6:1混合,将此蛋白粕混合物与水按质量体积比1:4~14混匀,室温搅拌1.5~2.5h后置于超高压主机***中,处理压力200~600MPa,保压时间10~30min,获得经超高压预处理的混悬液;将混悬液控温至40~60℃,调pH至9~10,加入混悬液质量2%~6%的碱性蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200~400W,超声时间10~20min,微波功率200~600W,微波时间5~15min,酶解1.5~2.5h后调pH至6~8,加入混悬液质量2%~6%的木瓜蛋白酶并搅拌均匀,同时超声波-微波辅助酶解,超声功率200~400W,超声时间10~20min,微波功率200~600W,微波时间5~15min,酶解2~3h后升温灭酶,板框压滤后收集清液。
3.根据权利要求1所述的制备方法,其特征在于,步骤(3)中,将洗净的盖玻片浸泡于1.5~10g/L高精氨酸混合肽水溶液中,20~40rpm振荡4~8h后,纯水漂洗晾干,获得混合肽固定化模板。
4.根据权利要求1所述的制备方法,其特征在于,步骤(3)中,将功能单体丙烯酸、甲基丙烯酸,交联剂三乙二醇二甲基丙烯酸酯按体积比0.5~3.5:0.5~2.5:4~11混匀,加入0.2~0.8体积1~4mmol/L的异丙基硫杂蒽酮的丙酮溶液作为光引发剂,获得预聚混合液。
5.根据权利要求1所述的制备方法,其特征在于,步骤(3)中,向预聚混合液中通入氮气20~40min后,将其平铺在固定于旋转机上的硅烷化载玻片表面,旋转机100~400rpm旋转2~10s后,将混合肽固定化模板覆盖于载玻片上,365nm紫外光引发聚合3~6h。
6.根据权利要求1所述的制备方法,其特征在于,步骤(4)中,高精氨酸混合肽表面印迹膜重复吸附-洗脱过程10次以上。
7.一种高精氨酸混合肽,其特征在于,通过权利要求1至6中任一项所述的制备方法制备。
8.根据权利要求7所述的高精氨酸混合肽,其特征在于,所述混合肽中精氨酸含量在18%以上。
9.根据权利要求7所述的高精氨酸混合肽在制备与***治疗相关的药品中的应用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610520561.3A CN107574209B (zh) | 2016-07-05 | 2016-07-05 | 一种高精氨酸混合肽的制备方法及其在***治疗中的应用 |
US15/642,278 US20180009842A1 (en) | 2016-07-05 | 2017-07-05 | Arginine-rich Peptide Mixture, their Application Thereof in Cervical Cancer Therapy, and a Process for Producing same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610520561.3A CN107574209B (zh) | 2016-07-05 | 2016-07-05 | 一种高精氨酸混合肽的制备方法及其在***治疗中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107574209A CN107574209A (zh) | 2018-01-12 |
CN107574209B true CN107574209B (zh) | 2021-02-05 |
Family
ID=60893172
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610520561.3A Active CN107574209B (zh) | 2016-07-05 | 2016-07-05 | 一种高精氨酸混合肽的制备方法及其在***治疗中的应用 |
Country Status (2)
Country | Link |
---|---|
US (1) | US20180009842A1 (zh) |
CN (1) | CN107574209B (zh) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107583031A (zh) * | 2016-07-06 | 2018-01-16 | 陈栋梁 | 一种清蛋白肽混合物的制备方法及其抑制癌细胞增殖作用 |
CN108893518A (zh) * | 2018-07-12 | 2018-11-27 | 吉首大学 | 一种能抑制人***细胞增殖的药物及检测方法 |
CN110354826B (zh) * | 2019-07-29 | 2021-12-14 | 肇庆学院 | 一种铅镉离子双模板磁性分子印迹聚合物及其制备方法 |
CN110763539B (zh) * | 2019-11-22 | 2021-08-31 | 福州大学 | 一种基于分子印迹柱和重量体系的羟基多氯联苯检测方法 |
CN111389234B (zh) * | 2020-03-20 | 2022-03-22 | 江苏大学 | 一种三维多孔MnO2纳米线印迹膜及其制备方法与应用 |
CN112795611B (zh) * | 2021-01-25 | 2023-04-28 | 昆明生物制造研究院有限公司 | 一种不可溶性蛋白制备核桃蛋白多肽的方法 |
CN113854398A (zh) * | 2021-10-08 | 2021-12-31 | 长春大学 | 一种核桃饼粕为原料制备免消化核桃蛋白质的方法 |
WO2024122437A1 (ja) * | 2022-12-04 | 2024-06-13 | 国立大学法人 熊本大学 | アミノ酸分析システム及びアミノ酸分析方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101775103A (zh) * | 2009-12-29 | 2010-07-14 | 湖北工业大学 | 一种蛋白质分子印迹薄膜的制备方法 |
CN103109971A (zh) * | 2013-03-04 | 2013-05-22 | 四川省均易润泽食品有限公司 | 制备高精氨酸核桃肽的方法 |
-
2016
- 2016-07-05 CN CN201610520561.3A patent/CN107574209B/zh active Active
-
2017
- 2017-07-05 US US15/642,278 patent/US20180009842A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101775103A (zh) * | 2009-12-29 | 2010-07-14 | 湖北工业大学 | 一种蛋白质分子印迹薄膜的制备方法 |
CN103109971A (zh) * | 2013-03-04 | 2013-05-22 | 四川省均易润泽食品有限公司 | 制备高精氨酸核桃肽的方法 |
Non-Patent Citations (6)
Title |
---|
Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents-A review;E. D. N. S. Abeyrathne等;《Poultry Science》;20131201;第92卷(第12期);第3292页第1段摘要部分 * |
Incorporation of styrene enhances recognition of ribonuclease A by molecularly imprinted polymers;Chung-Yi Hsu等;《Biosensors and Bioelectronics》;20060614(第22期);第356页右栏以及"Scheme 1"部分 * |
Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells;Sihui Ma等;《BMC Complementary and Alternative Medicine》;20151123;第2页左栏-第3页右栏、第7页左栏第1段 * |
Towards molecularly imprinted polymers selective to peptides and proteins. The epitope approach;Alexandre Rachkov等;《Biochimica et Biophysica Acta》;20010109;第1544卷(第1期);第255-266页 * |
低分子质量核桃多肽抗氧化及抗肿瘤活性研究;许典等;《中国油粮学报》;20151125;第30卷(第11期);第65-75页 * |
核桃仁蛋白木瓜酶水解物抑制癌细胞增殖;翟梦新等;《食品科技》;20130920;第38卷(第09期);第6页摘要部分 * |
Also Published As
Publication number | Publication date |
---|---|
US20180009842A1 (en) | 2018-01-11 |
CN107574209A (zh) | 2018-01-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107574209B (zh) | 一种高精氨酸混合肽的制备方法及其在***治疗中的应用 | |
CN104926925B (zh) | 一种带鱼鱼肉蛋白抗氧化肽及其制备方法和用途 | |
CN103626847B (zh) | 一种小麦胚芽蛋白源锌螯合肽及其制备方法 | |
CN103230587B (zh) | 一种鳄鱼血红蛋白肽的组合物及其制备方法 | |
CN104762358B (zh) | 贻贝蛋白降压肽的快速制备方法 | |
CN103275181B (zh) | 一种金枪鱼碎肉多肽类血管生成抑制因子及其制备方法和用途 | |
CN105624251B (zh) | 一种牡丹籽降血糖肽及其纯化方法和应用 | |
CN104774899B (zh) | 金枪鱼暗色肉蛋白抗***多肽的制备方法 | |
CN111518164B (zh) | Ace抑制肽p2、其应用及其制备方法 | |
CN107574207B (zh) | 一种高精氨酸混合肽的制备方法及其在结直肠癌治疗中的应用 | |
CN107574210B (zh) | 一种高精氨酸混合肽的制备方法及其在肝癌治疗中的应用 | |
CN1985844A (zh) | 磁性吸附剂的制备方法及在治疗高胆红素血症的应用 | |
CN107574208B (zh) | 一种高精氨酸混合肽的制备方法及其在肺癌治疗中的应用 | |
CN107574206B (zh) | 一种高精氨酸混合肽的制备方法及其在乳腺癌治疗中的应用 | |
CN107574205B (zh) | 一种高精氨酸混合肽的制备方法及其在胃癌治疗中的应用 | |
DE3914869C1 (zh) | ||
CN106928344A (zh) | 用于从含有凝血因子的溶液中减少和/或除去FXI和FXIa的方法 | |
CN105648010B (zh) | 一种激活Nrf2-ARE通路的双髻鲨鱼肉抗氧化肽的制备方法 | |
CN104945471B (zh) | 贻贝蛋白降压肽 | |
CN111087447A (zh) | 一种鳄鱼抗氧化肽复合物及其制备方法和应用 | |
CN109369781A (zh) | 一种麒麟菜抗氧化四肽及其应用 | |
CN104757561A (zh) | 一种贻贝蛋白降压肽的用途 | |
CN104758925A (zh) | 带鱼鱼骨铁螯合胶原肽的铁螯合用途 | |
CN109248668A (zh) | 用于血液体外循环去除ldl的吸附剂及其制备方法和灌流器 | |
CN108410936A (zh) | 一种水牛乳抗氧化肽及其分离制备方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |