CN106191184A - A kind of preparation of novel Scapharca broughtonii antioxidation active peptides and application thereof - Google Patents
A kind of preparation of novel Scapharca broughtonii antioxidation active peptides and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of novelty Scapharca broughtonii (Arcainflata Reeve) the separation preparation and application thereof of antioxidation active peptides.Described method for separating and preparing includes shelling Scapharca broughtonii cleaning, tissue homogenate, hydrolysis process screening and optimizing, and centrifuging and taking supernatant lyophilizing obtains active polypeptide component；Active component is carried out the most successively ultrafiltration and retains concentration, ion-exchange chromatography, gel filtration and efficient exclusion chromatography, finally give the antioxidant activity polypeptide that purity is 99%.The present invention creatively use various modern Protein purification techniques from Scapharca broughtonii hydrolysate one material base of isolated clearly, molecular weight be 17578.31Da, containing secondary structure alpha-helix 40.8%, beta sheet 24.7%, β-corner 15.9%, the active polypeptide of random coil 18.6%.This polypeptide has significant antioxidant activity, external removing DPPH, ABTS and hydroxyl radical free radical IC50Value is respectively 3.89,0.22 and 10.77mg/ml, and Caenorhabditis elegans average life can be made to extend 18.29%.Therefore can be used for the development and application of antioxidant, cosmeceutical or health product.
Description
Technical field
The invention belongs to biomedicine technical field, relate to the antioxidant activity polypeptide compound in a kind of marine organisms Scapharca broughtonii (Arca inflata Reeve), and method for separating and preparing and the antioxidation medical usage of this compound.
Background technology
Scapharca broughtonii (Arca inflata Reeve) belongs to animal door, lamellibranchiata, Carnis Arca inflata mesh, Carnis Arca inflata section, belong to a member of sea mollusk door, it it is economic shellfish of dwelling at the bottom of a kind of large ocean, it is distributed mainly on Japan, the Korea peninsula and the Bohai Sea of China, bottom the ooze of the Huanghai Sea and East China Sea lefteye, one of economic shellfish that Ye Shi China is important, its delicious meat, blood is scarlet, there is high protein, low fat, the features such as vitamin is abundant, the most just by people as excellent tonic product, go well with wine famous dish, Scapharca broughtonii described in ancient book has and " makes us eating, benefit color, disappear clot and reduce phlegm long-pending " effect.
Finding by consulting data of literatures, foreign scholar elaborates that the class in Scapharca broughtonii has the protein polypeptide active component of antibacterial action and immunoregulation effect, and the research that extraction has anti-oxidation active substance has no report.Domestic scholars focuses primarily upon the aspects such as Scapharca broughtonii ecological habit, cultural technique and Analysis of Nutritive Composition thereof to the research of Scapharca broughtonii, also little, as Chinese patent 200610036488.9 discloses " polypeptide protein class active substance in Scapharca broughtonii and its production and use " to Scapharca broughtonii protein and peptide thing Quality Research；Patent 201310619247.7 discloses " separating method and the purposes preparing a kind of antineoplastic polypeptide compound from Scapharca broughtonii ", they all describe method and the antineoplastic medical usage thereof extracting activated protein polypeptides matter from Scapharca broughtonii natural product in detail, but rare have to extract from Scapharca broughtonii hydrolysate there is the activated protein polypeptide fractions of antioxidation, anti-aging effects, also have no that any extraction from Scapharca broughtonii hydrolysate is separated to high-purity and the clear and definite antioxidant activity polypeptide compound of material base and physicochemical property, the research report of structure elucidation.Therefore utilize the active substance hydrolyzed in this method extraction Scapharca broughtonii to expand the separation method of active polypeptide component in Scapharca broughtonii further, also enrich the extraction approach of activity polypeptid substance in other species, provide scientific basis for Appropriate application marine resources；Resolving significant additionally, polypeptide compound carries out deep property testing and basic structure, the pharmacological mechanism for material is provided fundamental basis.
Summary of the invention
It is an object of the invention to by various modern separation and purification of protein technology, extract from marine organisms Scapharca broughtonii hydrolysate and separate protein and peptide active substance, a kind of Scapharca broughtonii hydrolysis process optimized particularly is provided, and relate to the contents such as its preparation method, medical usage and structural characterization.
The present invention is to be realized by following technical method: cleaning of being shelled by fresh Scapharca broughtonii, tissue homogenate, and hydrolysis process screening and optimization, centrifuging and taking supernatant is dried concentration, obtains active polypeptide component.Active component carrying out ultrafiltration the most successively and retains concentration, ion-exchange chromatography, gel filtration and efficient exclusion chromatography, finally give the antioxidant activity polypeptide that purity is 99%, molecular weight is 17578.31 Da, and resolves its basic structure.
Antioxidation in vitro test result indicate that, this polypeptide compound has significant antioxidant activity, external removing DPPH, ABTS and hydroxyl radical free radical IC50Value is respectively 3.89,0.22 and 10.77 mg/ml；Internal antioxidation experimental result shows, Caenorhabditis elegans MaLS can be made to extend to 27 days, and average life increases by 18.29%.Therefore this polypeptide compound can be used for the development and application of antioxidant, cosmeceutical or health product.
Accompanying drawing explanation
Figure1 is the relative molecular weight measuring active polypeptide compound in example 1 with matrix solid-dispersion time-of-flight mass spectrometryFigureSpectrum.
Figure2 is the circular dichroism spectra of polypeptide compound in example 1Figure。
Figure3 utilize matrix solid-dispersion time-of-flight mass spectrometry to measure the fingerprint of active polypeptide for polypeptide compound in example 1FigureSpectrum.
Detailed description of the invention
Below in conjunction with technical scheme andFigureTable describes the specific embodiment of the present invention in detail.
Embodiment
1
Select a kind of commercial protease that Scapharca broughtonii tissue is hydrolyzed, utilize modern separation and purification of protein technology, carry out active polypeptide component in Scapharca broughtonii separating preparation.
Scapharca broughtonii is peeled off, take its tissue agglomerate, distilled water cleans three times, weigh after draining, according to 1:3(w/v) ratio add distilled water, use high-speed tissue mashing machine, with 3000 rpm, it is homogenized 1 min at interval of 30sec, amount to 3min, homogenate is placed in ultrasonic 40 min in low frequency ultrasound instrument, add 5%(g/100g protein substrate) ratio neutral protease, maintain temperature 45 C, pH 7.0 reacts 5 h, 1M NaOH regulates pH, boiling water bath 10min terminates reaction, it is cooled to room temperature, then 8000 rpm, under the conditions of 4 DEG C, centrifugal 30 min go precipitation, take supernatant lyophilization；Utilizing the ultrafilter membrane centrifuge tube that molecular cut off is 3kD and 10kD in 4 DEG C, under the conditions of 3000 rpm, centrifugal 60 min, are three parts by active polypeptide component；Purpose bioactive peptide component crosses DEAE-Sephorose Fast Flow anion-exchange column, and be respectively adopted the pH8.0 Tris-HCl buffer containing 0,0.1,0.3,0.5,1.0 M NaCl and carry out step gradient, flow velocity is 1 ml/min, detects absworption peak at 280 nm；Collect the polypeptide fractions under 0.3 M NaCl eluting, cross Sephadex-G100 sephadex column, 0.3 ml/min ultra-pure water eluting, at 280 nm, detect absworption peak；Collect Sephadex-G100 gel column isolating active polypeptide fractions, utilize liquid size-exclusion method, use TSK-Gel G2000SWxl chromatographic column, with containing 0.1M Na2SO4, the PBS buffer solution of the 0.05M of pH 6.8 is flowing phase, flow velocity 1 ml/min；Detection wavelength 280 nm, separates the active polypeptide preparing a kind of high purity 99%.
Using MALDI-TOF/TOF-MS(matrix solid-dispersion time-of-flight mass spectrometry) to measure the molecular weight of this polypeptide be 17578.31 Da to method, and measure the fingerprint of polypeptideFigureSpectrum；Its secondary structure composition is analyzed by circular dichroism spectra and Jasco Protein secondary structure assessment software.
Table
1. the secondary structure composition analysis result of active polypeptide compoundTable 1
| Secondary structure type | Composition (%) |
| Alpha-helix | 40.8 |
| Beta sheet | 24.7 |
| β-corner | 15.9 |
| Random coil | 18.6 |
Embodiment
2
Use three kinds of external free radical scavenging experiments, active polypeptide compound in Scapharca broughtonii hydrolysate is carried out antioxidant activity analysis.
(1) DPPH free radical scavenging activity measures: distilled water compound concentration is the sample solution of 1,2,4 and 8 mg/ml, take sample solution and the DPPH solution of 190 μ L 0.2 mM of 10 μ L variable concentrations, add in 96 well culture plates, lucifuge is room temperature reaction 30 min on horizontal shaker, its absorbance is measured at microplate reader 517 nm wavelength, each concentration sets three groups of parallel tests, and result takes its meansigma methods, and reduced glutathion is positive control.
(2) ABTS free radical scavenging activity measures: distilled water compound concentration is the sample solution of 1,2,4 and 8 mg/ml, take sample solution and the ABTS free radical working solution of 190 μ L 10 mM pH7.4 of 10 μ L variable concentrations, add in 96 well culture plates, lucifuge is room temperature reaction 10 min on horizontal shaker, measure its absorbance at microplate reader 734 nm wavelength, test set 4 groups parallel, results averaged, and repeat to test three times, reduced glutathion is positive control.
(3) hydroxyl radical free radical scavenging capacity measures: prepare the sodium phosphate buffer (PBS) of 0.1 mol/L PH 7.4,5.0 mmol/L orthophenanthroline solution, the FeSO of 5.0 mmol/L respectively4Solution, 0.3% H2O2Solution and the sample solution of 1,2,4 and 8 mg/ml, in 96 orifice plates according toTable 3It is sequentially added into corresponding solution, reaction cumulative volume is 200 μ L, above-mentioned each group of example reaction system is shaken up and is placed in constant incubator, 37 DEG C of reaction 60 min, then absorbance (A) is measured at microplate reader 510 nm wavelength, at least setting 3 groups of parallel tests, results averaged, glutathion is positive control.
Table 3. hydroxyl radical free radical scavenging capacity measures operating procedure
Table 4. three kinds of external free radical scavenging measuring results
Embodiment
3
Use model animal Caenorhabditis elegans, carry out the internal anti-aging effects analysis of active polypeptide.
Selecting synchronized L4 phase C. Elegans Automatic Screening to be administered, dosage is respectively 2,4 and 8 mg/ml, cultivates 48 h under the conditions of 20 DEG C, and picking matched group and experimental group nematicide are to scribbling respectivelyE.coliThe fresh NGM culture dish of OP50 is cultivated, the C. Elegans Automatic Screening of survival is transferred in fresh NGM culture dish by every day, and the death toll of accurate recording C. Elegans Automatic Screening on the lower same day and lose number, until there is no the C. Elegans Automatic Screening lived, confirm that the standard of its death is that nematicide can not produce responsing reaction, blank group and the experimental group the last item C. Elegans Automatic Screening dead time and be the MaLS of this group nematicide outside stimulus；The nematicide number at least repeating parallel laboratory test twice, matched group and experimental group is 50 ± 2.
Table 5. polypeptide compound to the aging effects of Caenorhabditis elegans (± S, n=3, * p < 0.05)
Claims (10)
1. Scapharca broughtonii (Arca inflata Reeve) antioxidant activity polypeptide compound, carry out separating preparation by the following method: peeled off by fresh Scapharca broughtonii, take out content cleaning, tissue homogenate processes, add protease hydrolysis, centrifuging and taking supernatant, carry out ultrafiltration after lyophilization successively and retain concentration, ion-exchange chromatography, gel filtration and efficiently exclusion chromatography separation, finally give the active polypeptide compound that purity is 99%.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, it is characterised in that tissue homogenate processes, and adds distilled water volume and Scapharca broughtonii tissue weight ratio for 1:3, and tissue mashing machine is homogenized, and speed is 3000
Rpm, the time is 3 min, per minute between rest 30 seconds.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, it is characterised in that design four factor three horizontal quadrature test (L934), use neutral protease to be hydrolyzed, four levels are: ratio at the bottom of pH value, temperature, time, enzyme, three levels of pH value are: 6.0,7.0,8.0, three levels of temperature are 45,50,55 DEG C, and three levels of time are 4,5,6 h, and three levels of ratio at the bottom of enzyme are 3%, 4%, 5%.
4. prepare according to the separation of antioxidant activity polypeptide compound in the Scapharca broughtonii described in claim 1 and 3, it is characterized in that using neutral protease to be hydrolyzed, the hydrolysising condition finally determined is pH 7.0, temperature 45 C, time 5 h, ratio 5% at the bottom of enzyme, 1M NaOH regulates pH, and boiling water bath boils 10 min and terminates reaction, in 4 DEG C after cooling, 8000 rpm are centrifuged 30min and go precipitation, take supernatant lyophilization.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, it is characterized in that, by after active polypeptide component lyophilizing, utilizing the ultrafilter membrane centrifuge tube that molecular cut off is 3kD and 10kD that polypeptide fractions is divided into three parts, centrifugal condition is 4 DEG C, 3000 rpm, 60 min.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, after it is characterized in that retaining active polypeptide component ultrafiltration, DEAE-Sephorose Fast Flow anion-exchange column is utilized to separate, the pH8.0 Tris-HCl buffer containing 0,0.1,0.3,0.5,1.0 M NaCl is used to carry out step gradient successively, 1 ml/min, detects absworption peak at 280 nm.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, it is characterized in that collecting the polypeptide fractions under 0.3 M NaCl eluting, separate with Sephadex-G100 sephadex column, eluent is ultra-pure water, flow velocity is 0.3 ml/min, detects absworption peak at 280 nm.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, it is characterised in that
Collect Sephadex-G100 gel column isolating active polypeptide fractions, utilize liquid size-exclusion method, use TSK-Gel
G2000SWxl chromatographic column, with containing 0.1M Na2SO4, the PBS buffer solution of pH 6.8 is flowing phase, flow velocity 1 ml/min；Detection wavelength 280 nm, separates the active polypeptide preparing a kind of high purity 99%.
In Scapharca broughtonii the most according to claim 1 prepared by the separation of antioxidant activity polypeptide compound, the molecular weight that it is characterized in that the Scapharca broughtonii polypeptide compound obtained is 17578.31Da, isoelectric point, IP 5.78, containing secondary structure alpha-helix 40.8%, beta sheet 24.7%, β-corner 15.9%, the active polypeptide of random coil 18.6%.
10. in the Scapharca broughtonii described in claim 1, antioxidation polypeptide compound can extend the life-span of model animal Caenorhabditis elegans, also can be used for the development and application of antioxidant, cosmeceutical or health product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109402090A (en) * | 2018-11-14 | 2019-03-01 | 于荣敏 | A kind of β -1,3 endoglucanase and its coded polynucleotide with immune-enhancing activity in chief blood clam source |
| CN112724198A (en) * | 2019-10-28 | 2021-04-30 | 于荣敏 | Methicillin-resistant staphylococcus aureus-resistant antibacterial peptide and preparation method and application thereof |
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| EP1593685A1 (en) * | 2004-05-04 | 2005-11-09 | Kraft Foods Holdings, Inc. | Peptide antioxidants from soy protein |
| CN101906456A (en) * | 2010-07-22 | 2010-12-08 | 浙江工商大学 | Preparation method and use of antioxidant peptide derived from mytilus coruscus |
| CN102372765A (en) * | 2010-08-05 | 2012-03-14 | 浙江海洋学院 | Ruditapes philippinarum enzymatic polypeptide and preparation method thereof |
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| EP1593685A1 (en) * | 2004-05-04 | 2005-11-09 | Kraft Foods Holdings, Inc. | Peptide antioxidants from soy protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402090A (en) * | 2018-11-14 | 2019-03-01 | 于荣敏 | A kind of β -1,3 endoglucanase and its coded polynucleotide with immune-enhancing activity in chief blood clam source |
| CN109402090B (en) * | 2018-11-14 | 2022-10-21 | 于荣敏 | Beta-1,3 endoglucanase with immune enhancing activity and derived from scapharca broughtonii and encoding polynucleotide thereof |
| CN112724198A (en) * | 2019-10-28 | 2021-04-30 | 于荣敏 | Methicillin-resistant staphylococcus aureus-resistant antibacterial peptide and preparation method and application thereof |
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