CN105646657A - Sphyrna lewini chondroprotein antioxidative peptide and application thereof - Google Patents
Sphyrna lewini chondroprotein antioxidative peptide and application thereof Download PDFInfo
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- CN105646657A CN105646657A CN201511014862.0A CN201511014862A CN105646657A CN 105646657 A CN105646657 A CN 105646657A CN 201511014862 A CN201511014862 A CN 201511014862A CN 105646657 A CN105646657 A CN 105646657A
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Abstract
The invention discloses a preparation method of sphyrna lewini chondroprotein antioxidative peptide. In the method, sphyrna lewini cartilage is taken as a raw material; an enzymatic hydrolysate is obtained through total protein extraction, acetone fractional precipitation, and trypsin enzymatic hydrolysis; 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 Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly. The molecular weight of the antioxidative peptide is measured through ESI-MS and is 950.03 Da. The prepared high-activity antioxidative peptide has high free-radical scavenging activity and an excellent lipid-peroxidation inhibiting effect, and can be used for developing a medicine, a health-care food, and a food additive.
Description
Technical field
The present invention relates to a kind of antioxidation active peptides, be specifically related to a kind of hammerhead chondroprotein anti-oxidation peptide and application thereof.
Background technology
Shark whole body is precious, and some kind delicious meat, are dish common on dining table. Fish skin has good toughness, is the raw material of leather processing industries. Modern medicine proves that shark cartilage has very big medical value, and within 1992, first shark cartilage powder is directly used in clinical treatment tumour by the U.S.. Hammerhead (Sphyrnalewini) is Chondrichthyes, Carcharhiniforme, and hammerhead section is distributed mainly on the Huanghai Sea, the East Sea and the South Sea in China.
Summary of the invention
The technical problem to be solved be to provide a kind of can the hammerhead 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 hammerhead chondroprotein anti-oxidation peptide: anti-oxidation peptide is decapeptide compound, aminoacid sequence is Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly, ESI-MS mensuration molecular weight is 950.03Da
The preparation method of this hammerhead chondroprotein anti-oxidation peptide is:
1) preparation of hammerhead cartilage total protein crude extract: weigh hammerhead cartilage and be cut into fine grained chippings, add appropriate distilled water, in high-speed tissue mashing machine, homogenate is to pasty state, the hammerhead 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 hammerhead cartilage total protein crude extract.
2) preparation of hammerhead cartilage acetone fractional precipitation albumen: taking hammerhead 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 hammerhead cartilage 60% acetone precipitation albumen, lyophilizing, be 60% acetone fractional precipitation albumen.
3) enzymolysis of hammerhead cartilage acetone fractional precipitation albumen: take hammerhead 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.
4) preparation of hammerhead chondroprotein anti-oxidation peptide: adopt 3kDa ultrafilter membrane to carry out hyperfiltration treatment above-mentioned enzymolysis solution, collects molecular weight less than 3kDa part, lyophilizing, obtains 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, the described hammerhead in step 1) is hammerhead (Sphyrnalewini).
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 Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) according to the scavenging capacity of DPPH free radical and hydroxyl radical free radical, it is 950.03Da 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 hammerhead 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, ABTS free radical and ultra-oxygen anion free radical are had good scavenging action by hammerhead chondroprotein anti-oxidation peptide provided by the present invention; Meanwhile, Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) also demonstrates good Lipid peroxidation;Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) 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 Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG).
The lipid peroxidation inhibition ability of Fig. 5 Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG).
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 a kind of hammerhead chondroprotein anti-oxidation peptide, preparation technology flow process is as follows: hammerhead 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.
1) preparation of hammerhead cartilage total protein crude extract: weigh hammerhead (Sphyrnalewini) cartilage and be cut into fine grained chippings, add appropriate distilled water, in high-speed tissue mashing machine, homogenate is to pasty state, the hammerhead cartilage of pasty state is dipped in the 1.0mol/L guanidine hydrochloride solution of 4 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 5000r/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 hammerhead cartilage total protein crude extract.
2) preparation of hammerhead cartilage acetone fractional precipitation albumen: take hammerhead 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, 10000r/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 hammerhead cartilage 60% acetone precipitation albumen, lyophilizing, obtains 60% acetone fractional precipitation albumen.
3) enzymolysis of hammerhead cartilage acetone fractional precipitation albumen: take hammerhead 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.
4) preparation of hammerhead chondroprotein anti-oxidation peptide: adopt 3kDa ultrafilter membrane to carry out hyperfiltration treatment above-mentioned enzymolysis solution, collect molecular weight less than 3kDa part (SP60-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: SP60-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 SP60-3(Fig. 1);
2. gel filtration chromatography: SP60-3 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 SP60-3-1(Fig. 2);
3. RP-HPLC purification: SP60-3-1 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 Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG), and it is 950.03Da([M+H that ESI-MS measures molecular weight]+951.24Da) (Fig. 4).
Prepared hammerhead chondroprotein anti-oxidation peptide Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) is carried out free radical scavenging experiment and lipid peroxidation Inhibition test, test result indicate that: Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) is to DPPH free radical (EC502.76mg/mL), hydroxyl radical free radical (EC500.22mg/mL), ABTS free radical (EC500.08mg/mL) with ultra-oxygen anion free radical (EC500.13mg/mL) there is good scavenging action; Meanwhile, Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG) 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>hammerhead chondroprotein anti-oxidation peptide and application thereof
<130>zjou-wb-201511-101
<160>1
<170>PatentInversion3.5
<210>1
<211>10
<212>PRT
<213>artificial sequence
<400>1
GlyPheThrGlyProProGlyPheAsnGly
1510
Claims (2)
1. hammerhead chondroprotein anti-oxidation peptide, it is characterised in that: this anti-oxidation peptide is decapeptide compound, and aminoacid sequence is Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly, ESI-MS mensuration molecular weight is 950.03Da.
2. the purposes of hammerhead chondroprotein anti-oxidation peptide, it is characterised in that: this anti-oxidation peptide is for preparing the purposes of antioxidant functional food or food or food additive.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113527422A (en) * | 2021-07-10 | 2021-10-22 | 浙江省农业科学院 | Polypeptide FLCQWP with anti-oxidation function and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007217358A (en) * | 2006-02-17 | 2007-08-30 | Nippon Meat Packers Inc | Anti-oxidizing peptide |
| CN104892729A (en) * | 2015-05-12 | 2015-09-09 | 浙江海洋学院 | Angiogenesis inhibitory factor for cartilage of Sphyrna lewini |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007217358A (en) * | 2006-02-17 | 2007-08-30 | Nippon Meat Packers Inc | Anti-oxidizing peptide |
| CN104892729A (en) * | 2015-05-12 | 2015-09-09 | 浙江海洋学院 | Angiogenesis inhibitory factor for cartilage of Sphyrna lewini |
Non-Patent Citations (2)
| 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》 * |
| 胡发远: "孔鳐软骨多肽及泥蚶多肽的制备及活性研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113527422A (en) * | 2021-07-10 | 2021-10-22 | 浙江省农业科学院 | Polypeptide FLCQWP with anti-oxidation function and application thereof |
| CN113527422B (en) * | 2021-07-10 | 2024-02-09 | 浙江省农业科学院 | Polypeptide FLCQWP with antioxidant function and application thereof |
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