CN105646651A - Dasyatis akajei chondroprotein antioxidative peptide and application thereof - Google Patents
Dasyatis akajei chondroprotein antioxidative peptide and application thereof Download PDFInfo
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- CN105646651A CN105646651A CN201511014853.1A CN201511014853A CN105646651A CN 105646651 A CN105646651 A CN 105646651A CN 201511014853 A CN201511014853 A CN 201511014853A CN 105646651 A CN105646651 A CN 105646651A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 31
- 230000003078 antioxidant effect Effects 0.000 title abstract description 9
- 241001430386 Dasyatis akajei Species 0.000 title abstract description 6
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 claims abstract description 5
- 235000013402 health food Nutrition 0.000 claims abstract description 4
- 235000013373 food additive Nutrition 0.000 claims abstract 2
- 239000002778 food additive Substances 0.000 claims abstract 2
- 230000003064 anti-oxidating effect Effects 0.000 claims description 24
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 abstract description 44
- 210000000845 cartilage Anatomy 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 15
- 238000001556 precipitation Methods 0.000 abstract description 14
- 238000004007 reversed phase HPLC Methods 0.000 abstract description 14
- 230000002000 scavenging effect Effects 0.000 abstract description 8
- 238000000108 ultra-filtration Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 6
- 230000003859 lipid peroxidation Effects 0.000 abstract description 5
- 102000004142 Trypsin Human genes 0.000 abstract description 4
- 108090000631 Trypsin Proteins 0.000 abstract description 4
- 239000012588 trypsin Substances 0.000 abstract description 4
- 238000004440 column chromatography Methods 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract 2
- 239000000413 hydrolysate Substances 0.000 abstract 2
- 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 abstract 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 239000003456 ion exchange resin Substances 0.000 abstract 1
- 229920003303 ion-exchange polymer Polymers 0.000 abstract 1
- 238000000751 protein extraction Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- -1 DPPH free radical Chemical class 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 239000012153 distilled water Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000000287 crude extract Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000001641 gel filtration chromatography Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 238000004255 ion exchange chromatography Methods 0.000 description 4
- 235000011837 pasties Nutrition 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 238000005728 strengthening Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000039233 Okamejei kenojei Species 0.000 description 2
- 230000002292 Radical scavenging effect Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000007096 poisonous effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 235000015961 tonic Nutrition 0.000 description 2
- 230000001256 tonic effect Effects 0.000 description 2
- 229960000716 tonics Drugs 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 206010009168 Chyluria Diseases 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 208000015817 Infant Nutrition disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000230053 Myliobatiformes Species 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241001290266 Sciaenops ocellatus Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 235000020989 red meat Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a dasyatis akajei chondroprotein antioxidative peptide and an application thereof. Dasyatis akajei cartilage is taken as a raw material; an enzymatic hydrolysate is obtained through total protein extraction, acetone fractional precipitation, and trypsin enzymolysis; and the enzymatic hydrolysate is separated and purified through ultrafiltration, ion exchange resin chromatography, gel column chromatography, and reversed-phase high-performance liquid chromatography to obtain the antioxidative peptide Ile-Glu-Pro-His having a molecular weight of 494.55 Da measured through ESI-MS. The prepared high-activity antioxidative peptide has an excellent scavenging effect on DPPH free radicals, hydroxyl free radicals, ABTS free radicals, and superoxide anionic free radicals and has an excellent inhibiting effect on lipid peroxidation. The antioxidative peptide is safe, has no toxic or side effect, and is high in antioxidant activity and easy to absorb and digest. The antioxidant activity can be used as a medicine, a health food, or a food additive.
Description
Technical field
The present invention relates to a kind of animal cartilage protein antioxidant peptide, be specifically related to a kind of red chondroprotein anti-oxidation peptide and application thereof.
Background technology
Red (Dasyatisakajei), belongs to Chondrichthyes, lower opening catalogue, Myliobatiformes, section, belongs to. Red for common cartilaginous fish, it is distributed in China coast, also sees the Xijiang River, until Nanning, Longzhou, Guiping, and Japan, the Korea peninsula.
Red for the conventional marine drug of China, book on Chinese herbal medicine is practised and claims Carnis Dasyatis akajei, begin to be loaded in supplement to the Herbal. Red whole body is precious, and its meat, tooth, anal spine etc. all can be used as medicine. Red meat has nourishing YIN and supplementing blood, and kidney tonifying is treating stranguria. Cure mainly dysuria, puckery pain, man's nebulousurine stranguria marked by chyluria, infantile malnutrition. Red tooth has the effects such as parasite killing, preventing the attack (or recurrence) of malaria, removing toxic substances, it is possible to the diseases such as treatment miasma is cruel, pruritus. Red liver has the effects such as nourishing the liver to improve visual acuity, strengthening by means of tonics, bone strengthening. Red anal spine is poisonous, has the effects such as heat-clearing and toxic substances removing, pain relieving, hard masses softening and resolving. Red fish oil is slightly poisonous, has the effects such as strengthening by means of tonics, blood activating and fat reducing. And modern pharmacology also demonstrates that the red multiple significant pharmacologically active that has, red cartilage mucopolysaccharide have good anticoagulant active and effect for reducing blood fat, with existing anticoagulation medicine activity quite; Red anal spine 20% ethanol extraction can suppress the growth of mouse S 180 sarcoma, and the polypeptide that red cartilage extracts can substantially reduce the number of the blood capillary of Murine Hepatoma22 and the number of chick chorioallantoic membrane new vessels.
Summary of the invention
The technical problem to be solved be the present situation for prior art provide a kind of can the red chondroprotein anti-oxidation peptide of scavenging free radicals and anti-lipid peroxidation effect.
The present invention solves that the technical scheme that above-mentioned technical problem is taked is: a kind of red chondroprotein anti-oxidation peptide: this red chondroprotein anti-oxidation peptide, it is characterized in that this anti-oxidation peptide is tetrapeptides compound, aminoacid sequence is Ile-Glu-Pro-His (IEPH), ESI-MS mensuration molecular weight is 494.55Da.
The preparation method of this red chondroprotein anti-oxidation peptide is:
1) preparation of red cartilage total protein crude extract: weigh Raja porosa cartilage and be cut into fine grained chippings, add appropriate distilled water, in high-speed tissue mashing machine, homogenate is to pasty state, the red cartilage of pasty state is dipped in the 1.0mol/L guanidine hydrochloride solution of 4��6 times of volumes (containing 0.02mol/LMES and 0.02mol/LEDTA, pH7��8), at 4 DEG C, stir extracting 36��48h.Extracting solution with the centrifugal 15��25min of 4000��6000r/min, is abandoned residue and is collected supernatant at 4 DEG C; Supernatant continues, at 4 DEG C, the centrifugal 15��25min of 10000��12000r/min, to take supernatant and remove insoluble impurities with 0.22 ��m of filtering with microporous membrane. Filtrate being loaded in the bag filter that molecular retention amount is 8000, with Tris-HCl(pH7.6) buffer dialyses 24��48h at 4 DEG C, and in bag filter, solution is red cartilage total protein crude extract.
) preparation of red cartilage acetone fractional precipitation albumen: taking red cartilage total protein crude extract, to be slowly added to pre-cold acetone to acetone concentration under ice bath be 30%, after standing 4��6h at-20 DEG C, in 4 DEG C, 10000��12000r/min high speed centrifugation 20min, take supernatant, adding pre-cold acetone to the final acetone concentration of solution is 60%, obtain red cartilage 60% acetone precipitation albumen, lyophilizing, be 60% acetone fractional precipitation albumen.
) enzymolysis of red cartilage acetone fractional precipitation albumen: take red cartilage 60% acetone fractional precipitation albumen and be dissolved in Tris-HCl buffer (0.05mol/L according to solid-liquid ratio 1g:3��5mL, pH7.5��8.5), add trypsin 1.9 �� 10 according to the 2.0��3.0% of 60% acetone fractional precipitation protein by weight4U/g), in 40 DEG C of enzymolysis 3��5h, then in 90 DEG C, the work of 10min enzyme denaturing, enzymolysis solution, in 4 DEG C, the centrifugal 15��25min of 10000��12000r/min, is abandoned residue and is collected supernatant, obtain chondroprotein enzymolysis solution.
) preparation of red chondroprotein anti-oxidation peptide: adopt 3kDa ultrafilter membrane to carry out hyperfiltration treatment above-mentioned enzymolysis solution, collect molecular weight less than 3kDa part, lyophilizing, obtain ultrafiltration zymolyte; By ultrafiltration zymolyte time through resinbed analysis, gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) purification, obtain anti-oxidation peptide.
As preferably, red for red (Dasyatisakajei) in described step 1).
As preferably, the detailed process of the resinbed analysis of described step 4), gel filtration chromatography and RP-HPLC purification is:
Resinbed is analysed: above-mentioned ultrafiltration zymolyte is dissolved in distilled water and is made into the solution that concentration is 45��55mg/mL, post is analysed through DEAE-52 resinbed, eluting is carried out with water, 0.1mol/L, 0.5mol/L and 1.0mol/LNaCl solution, flow velocity is 0.6��0.8mL/min, the every 3min of wash solution collects a pipe and detects in 280nm, solution in test tube is merged by peak, compare the DPPH free radical at each peak and the scavenging capacity of hydroxyl radical free radical, select the strongest component lyophilizing of activity, be ion-exchange chromatography zymolyte;
Gel filtration chromatography: above-mentioned ion-exchange chromatography zymolyte is dissolved in distilled water and is made into the solution that concentration is 45��55mg/mL, through polydextran gel SephadexG-15 column chromatography for separation, eluting is carried out with distilled water, flow velocity is 0.6��0.8mL/min, the every 3min of wash solution collects a pipe and detects in 280nm, merges solution in test tube by peak, compares the DPPH free radical at each peak and the scavenging capacity of hydroxyl radical free radical, select the strongest component lyophilizing of activity, be gel chromatography zymolyte;
RP-HPLC purification: above-mentioned gel chromatography zymolyte distilled water is made into the solution of 80��100 �� g/mL, RP-HPLC is utilized to be purified, obtaining 1 high activity anti-oxidation peptide Ile-Glu-Pro-His (IEPH) according to the scavenging capacity of DPPH free radical and hydroxyl radical free radical, it is 494.55Da that ESI-MS measures molecular weight.
Further preferably, described RP-HPLC condition is: sample size 10��15 �� L; Chromatographic column ZorbaxSB-C18(250mm �� 4.6mm, 5 ��m); Mobile phase: water-acetonihile gradient elution (0��32min acetonitrile concentration is at the uniform velocity risen to 50% by 0); Elution speed 0.6 ~ 1.0mL/min; Ultraviolet detection wavelength 280nm.
The present invention also provides for the purposes of red chondroprotein anti-oxidation peptide: due to its there is safe without toxic side effect, antioxidant activity is strong and is prone to advantages such as digesting and assimilating, thus can as the additive of medicine, health food and food.
Compared with prior art, DPPH free radical, hydroxyl radical free radical and ultra-oxygen anion free radical are had good scavenging action by red chondroprotein anti-oxidation peptide provided by the present invention; Meanwhile, Ile-Glu-Pro-His (IEPH) also demonstrates good Lipid peroxidation; Ile-Glu-Pro-His (IEPH) has safe without toxic side effect, antioxidant activity is strong and is prone to advantages such as digesting and assimilating, it is possible to as the additive of medicine, health food and food.
Accompanying drawing explanation
Fig. 1 is the DEAE-52 resinbed analysis figure of the present invention.
Fig. 2 is the polydextran gel SephadexG-15 tomographic map of the present invention.
Fig. 3 polydextran gel SephadexG-15 prepares the RP-HPLC of zymolyte and analyzes.
The mass spectrum of Fig. 4 Ile-Glu-Pro-His (IEPH).
The lipid peroxidation inhibition ability of Fig. 5 Ile-Glu-Pro-His (IEPH).
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment:
The preparation method of red chondroprotein anti-oxidation peptide, preparation technology flow process is as follows: red cartilage " cartilage total protein " cartilage 60% acetone precipitation albumen " trypsin digestion " zymolyte " ultrafiltration " ion-exchange chromatography " gel permeation chromatography " high performance liquid chromatography prepares " anti-oxidation peptide.
) preparation of red cartilage total protein crude extract: weigh Raja porosa cartilage and be cut into fine grained chippings, add appropriate distilled water, in high-speed tissue mashing machine, homogenate is to pasty state, the red cartilage of pasty state is dipped in the 1.0mol/L guanidine hydrochloride solution of 5 times of volumes (containing 0.02mol/LMES and 0.02mol/LEDTA, pH7.0), at 4 DEG C, extracting 48h is stirred. Extracting solution with the centrifugal 20min of 4500r/min, is abandoned residue and is collected supernatant at 4 DEG C; Supernatant continues, at 4 DEG C, the centrifugal 20min of 12000r/min, to take supernatant and remove insoluble impurities with 0.22 ��m of filtering with microporous membrane. Filtrate being loaded in the bag filter that molecular retention amount is 8000, with Tris-HCl(pH7.6) buffer dialyses 48h at 4 DEG C, and in bag filter, solution is red cartilage total protein crude extract.
) preparation of red cartilage acetone fractional precipitation albumen: take red cartilage total protein crude extract, being slowly added to pre-cold acetone to acetone concentration under ice bath is 30%, after standing 5h at-20 DEG C, in 4 DEG C, 12000r/min high speed centrifugation 20min, taking supernatant, adding pre-cold acetone to the final acetone concentration of solution is 60%, it is thus achieved that red cartilage 60% acetone precipitation albumen, lyophilizing, obtains 60% acetone fractional precipitation albumen.
) enzymolysis of red cartilage acetone fractional precipitation albumen: take red cartilage 60% acetone fractional precipitation albumen and be dissolved in Tris-hydrochloride buffer (0.05mol/L according to solid-liquid ratio 1g:4mL, pH8.0), trypsin 1.9 �� 10 is added according to the 2.5% of 60% acetone fractional precipitation protein by weight4U/g), in 40 DEG C of enzymolysis 4h, then in 90 DEG C, the work of 10min enzyme denaturing, enzymolysis solution, in 4 DEG C, the centrifugal 20min of 12000r/min, is abandoned residue and is collected supernatant, obtain chondroprotein enzymolysis solution.
) preparation of red chondroprotein anti-oxidation peptide: adopt 3kDa ultrafilter membrane to carry out hyperfiltration treatment above-mentioned enzymolysis solution, collect molecular weight less than 3kDa part (DP60-I), obtain ultrafiltration enzymolysis solution, by ultrafiltration enzymolysis solution successively through resinbed analysis, gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) purification, obtain anti-oxidation peptide.
1. resinbed analysis: DP60-I is dissolved in distilled water and is made into the solution that concentration is 50mg/mL, post is analysed through DEAE-52 resinbed, eluting is carried out with water, 0.1mol/L, 0.5mol/L and 1.0mol/LNaCl solution, flow velocity is 0.6mL/min, the every 3min of wash solution collects a pipe and detects in 280nm, solution in test tube is merged by peak, compare DPPH free radical and the Hydroxyl radical-scavenging ability at each peak, select the strongest component lyophilizing of oxidation resistance, be ion-exchange chromatography zymolyte DP60-3(Fig. 1);
2. gel filtration chromatography: DP60-3 is dissolved in distilled water and is made into the solution that concentration is 50mg/mL, through polydextran gel SephadexG-15 column chromatography for separation, eluting is carried out with distilled water, flow velocity is 0.6��0.8mL/min, the every 3min of wash solution collects a pipe and detects in 280nm, merges solution in test tube by peak, compares DPPH free radical and the Hydroxyl radical-scavenging ability at each peak, select the strongest component lyophilizing of oxidation resistance, be gel chromatography zymolyte DP60-3-1(Fig. 2);
3. RP-HPLC purification: DP60-3-1 distilled water is made into the solution of 80��100 �� g/mL, (RP-HPLC condition is: sample size 15 �� L to utilize RP-HPLC to be purified; Chromatographic column ZorbaxSB-C18(250mm �� 4.6mm, 5 ��m); Mobile phase: water-acetonihile gradient elution (0��32min acetonitrile concentration is at the uniform velocity risen to 50% by 0); Elution speed 0.8mL/min; Ultraviolet detection wavelength 280nm), obtain 1 high activity anti-oxidation peptide DCPE-B(Fig. 3 according to the scavenging capacity of DPPH free radical and hydroxy radical).
4. structure detection: collect DPPH free radical and the highest anti-oxidation peptide DCPE-B of Scavenging activity on hydroxyl free radical, it is simple spike through RP-HPLC detection, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Ile-Glu-Pro-His (IEPH), and it is 494.55Da([M+H that ESI-MS measures molecular weight]+495.96Da) (Fig. 4).
Prepared red chondroprotein anti-oxidation peptide Ile-Glu-Pro-His (IEPH) is carried out free radical scavenging experiment and lipid peroxidation Inhibition test, test result indicate that: Ile-Glu-Pro-His (IEPH) is to DPPH free radical (EC502.42mg/mL), hydroxyl radical free radical (EC500.39mg/mL), ABTS free radical (EC500.11mg/mL) with ultra-oxygen anion free radical (EC500.17mg/mL); Meanwhile, Ile-Glu-Pro-His (IEPH) also demonstrates good Lipid peroxidation (Fig. 5).
Finally, in addition it is also necessary to be only the specific embodiment of the present invention it is noted that listed above. It is clear that the invention is not restricted to above example, it is also possible to there are many deformation. All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.
SEQUENCELISTING
<110>Oceanography Institute Of Zhejiang
<120>red chondroprotein anti-oxidation peptide and application thereof
<130>zjou-wb-201511-201
<160>1
<170>PatentInversion3.5
<210>1
<211>4
<212>PRT
<213>artificial sequence
<400>1
IleGluProHis
Claims (2)
1. red chondroprotein anti-oxidation peptide, it is characterised in that: this anti-oxidation peptide is tetrapeptides compound, and aminoacid sequence is Ile-Glu-Pro-His, ESI-MS mensuration molecular weight is 494.55Da.
2. the purposes of red chondroprotein anti-oxidation peptide, it is characterised in that: this anti-oxidation peptide is for preparing the application of health food or food additive.
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| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
| CN103524596A (en) * | 2012-07-03 | 2014-01-22 | 浙江海洋学院 | Antioxidative peptide of shark protein as well as preparation method and use thereof |
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|---|---|---|---|---|
| CN103524596A (en) * | 2012-07-03 | 2014-01-22 | 浙江海洋学院 | Antioxidative peptide of shark protein as well as preparation method and use thereof |
| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
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| Title |
|---|
| ZHONGRUI LI等: "Influence of average molecular weight on antioxidant and functional properties of cartilage collagen hydrolysates from Sphyrna lewini, Dasyatis akjei and Raja porosa", 《FOOD RESEARCH INTERNATIONAL》 * |
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| CN105646651B (en) | 2020-06-30 |
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