CN106868084A - The preparation method and application of Ruditapes philippinarum high activity natineoplaston - Google Patents
The preparation method and application of Ruditapes philippinarum high activity natineoplaston Download PDFInfo
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Abstract
The invention discloses the preparation method of Ruditapes philippinarum high activity natineoplaston, the step such as separate including enzymolysis, ultrafiltration, strong cat ion exchange column chromatographic isolation, gel column chromatography, and disclose the application of Ruditapes philippinarum natineoplaston that above-mentioned preparation method obtains on A549 non-small lung cancers are suppressed.The present invention prepares Ruditapes philippinarum natineoplaston using optimal enzymolysis and purifying process, and ultrafiltration obtains component of the molecular weight less than 5 KDa and isolated and purified through two steps, acquires high activity polypeptide, and integrated artistic is simple, it is easy to popularization and application.
Description
Technical field
Technical field is utilized the present invention relates to aquatic products processing, more particularly, to a kind of preparation of Ruditapes philippinarum natineoplaston
Method and application.
Background technology
Biologically active peptide is inactive specified protein fragment in protein sequence, and this specified protein fragment can pass through
Gastro-intestinal digestion or food processing are discharged from Parent Protease, and show various biological functional activities, are lived in body life
Played an important role in dynamic.Biologically active peptide is formed by two or more amino acid condensations, typically with 2~20
Amino acid residue, molecular weight is generally less than 6KDa.Architectural characteristic (amino acid is constituted and sequence) influence activity of biologically active peptide
The activity of peptide so that biologically active peptide shows multiple biological activities, such as it is anti-oxidant, antitumor, anti-hypertension, antibacterial and anti-
Blood coagulation etc..China seashells species is enriched, and yield is occupied first of the world, and 2014 annual productions are up to 1 341.67 × 104t.Ocean shellfish
Class meat fertilizer is tender, tasty, is that a kind of economic worth is high containing nutriments such as substantial amounts of protein, amino acid, polysaccharide
Marine organisms.In most cases, seashells are only processed to food, and single varieties, added value of product is low.With enzyme
Solution technology extracts the polypeptide with biological functional activity, higher value application seashells from seashells or its processing byproduct
Resource, can obtain more preferable economic benefit.In recent years, the research that biologically active peptide is obtained from seashells receives pass extensively
Note.
Ruditapes philippinarum (Ruditapes philippinarum), is commonly called as mottle clam, is distributed widely in China coast ground
Area, is one of big cultivated shellfish of China four, is a kind of economical rich in protein, fat, polysaccharide and the essential trace elements of the human body
The high seashells of value.There are some researches show Ruditapes philippinarum extract has multiple biological activities.It is relevant in recent years luxuriant and rich with fragrance
The research of rule guest clam son's extract focuses primarily upon the aspects such as anti-oxidant, strengthen immunity and hypotensive, and is ground to antineoplastic
Study carefully less.I disclosed in October, 2016 " research that Ruditapes philippinarum natineoplaston is prepared using enzyme process " (《Fishery is modern
Change》The 5th phase of volume 43), Ruditapes philippinarum polypeptide is obtained using zymolysis technique by studying, the polypeptide product for acquiring only exists
It is external high to the inhibiting rate of A549, but purity and activity be not high, and only active peptide could be fine to being produced in body
Effect, it is therefore necessary to further researched and developed, improve Ruditapes philippinarum natineoplaston activity.
The content of the invention
It is many to obtain high activity it is an object of the invention to provide a kind of preparation method of Ruditapes philippinarum natineoplaston
Peptide.
To achieve the above object, the present invention uses following technical scheme：
The preparation method of Ruditapes philippinarum high activity natineoplaston, comprises the following steps：
S1. digest, take fresh Ruditapes philippinarum, after seawater tells sand, after being put into refrigerator freezing, open shell and take meat, be homogenized, feed liquid
Than 1:1, pH value 2.0 is adjusted, pepsin is added, enzyme concentration is 500~3000U/g, 31~43 DEG C of hydrolysis temperature, enzymolysis time
2~12h, is centrifuged after the enzyme that goes out, and takes supernatant liquor；
S2. ultrafiltration, the supernatant liquor that step S1 is obtained carries out ultrafiltration retention, collects the retention of below molecular weight 5KDa
Liquid, nanofiltration concentrating and desalinating, freeze-drying；
S3. strong cat ion exchange column chromatographic isolation, the product of freeze-drying is delayed by strong cat ion exchange column in step S2
After fliud flushing linear elution, detected at ultraviolet-visible spectrophotometer 280nm, each eluting peak is collected respectively, and dialysis is removed
Salt, freeze-drying；Dried each component is carried out into antitumor activity analysis, the eluting peak where further determining that target peptide,
And the elution fraction is largely collected, freeze-drying；
S4. gel column chromatography separate, by the product after freeze-drying in step S3 cross gel column wash-out, then it is ultraviolet-
Detected at visible spectrophotometer 280nm, each eluting peak is collected respectively, desalination of dialysing, freeze-drying will be dried each
Component carries out antitumor activity analysis, the eluting peak where further determining that target peptide, and the elution fraction is largely collected, cold
Jelly is drying to obtain Ruditapes philippinarum high activity natineoplaston.
Wherein, in step S3 use SP Sephadex C-25 strong cat ion exchange columns, buffer solution be containing concentration less than etc.
Receive in the acetic acid of 1mol/L NaCl-acetate buffer carries out linear elution, elution speed is 0.5~1.5mL/min.
Wherein, Sephadex G-25 gel columns are used in step S4, mobile phase is eluted for ultra-pure water, elution speed
It is 0.5~1.5mL/min.
Preferably, antitumor activity analysis uses CCK-8 methods in step S3 and S4.
Preferably, the enzyme concentration of pepsin is 1000U/g in step S1, and hydrolysis temperature is 37 DEG C, and enzymolysis time is 6h.
The invention also discloses the Ruditapes philippinarum that the preparation method of above-mentioned Ruditapes philippinarum high activity natineoplaston is obtained
Application of the natineoplaston on A549 non-small lung cancers are suppressed.
The present invention has the advantages that：Ruditapes philippinarum natineoplaston is prepared with purifying process using optimal enzymolysis,
Ultrafiltration obtains component of the molecular weight less than 5KDa and is isolated and purified through two steps, and acquisition can obtain high activity polypeptide.By the mesh of gained
Mark peptide is applied on A549 non-small lung cancers cells to generate very strong cell inhibitory effect effect, and demonstrate molecular weight 5KDa with
Under ultrafiltration retention component very strong tumor inhibition effect is generated on A549 tumor-bearing mices, be Ruditapes philippinarum natineoplaston
Preparation research provides test basis；Integrated artistic of the present invention is simple, and less input, it is easy to popularization and application.
Brief description of the drawings
Fig. 1 is block diagram of five kinds of enzymolysis products of protease to A549 non-small lung cancers cell proliferation inhibition rates.
Fig. 2 is block diagram of the pepsin different molecular weight enzymolysis product to A549 non-small lung cancers cell proliferation inhibition rates.
Fig. 3 is the SP Sephadex C-25 chromatography collection of illustrative plates of the component of below pepsin enzymolysis product molecular weight 5k.
Fig. 4 is that SP Sephadex C-25 strong cat ion exchange columns separation components press down to A549 non-small lung cancers cell propagation half
The block diagram of concentration (IC50) processed.
Fig. 5 is the Sephadex G-25 gel chromatography collection of illustrative plates at peak IV in Fig. 3.
Fig. 6 is that Sephadex G-25 gel columns separation components breed 503nhibiting concentration to A549 non-small lung cancers cell
(IC50) block diagram.
Fig. 7 is the tumour inhibiting rate block diagram of each group mouse.
Specific embodiment
In order that those skilled in the art more fully understands technical scheme, it is below in conjunction with the accompanying drawings and specific real
The present invention is described in further detail to apply example.
Process, and the product that will be prepared are analyzed in preparation present embodiment discloses Ruditapes philippinarum high activity natineoplaston
Thing carries out the process of mouse animal experiment, specific as follows：
1st, material prepares
(1) animal：Ruditapes philippinarum is purchased from certain supermarket, tells husky 1d, cleans, shells, taking meat, homogenate, and -20 DEG C of preservations are standby
With；SPF grades of Balb/c-nu grades of nude mouse, male and female half and half, 12~15g, 4~5 week old, 40, in Guangdong Province's medical experiment animal
The heart is provided.
(2) cell line：It is thin that A549 non-small lung cancers cells are purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine
Born of the same parents center.
(3) main agents：Pepsin, neutral proteinase, flavor protease, trypsase, alkali protease are purchased from
Pangbo Bioengineering Co Ltd, Nanning；SP Sephadex C-25 strong cat ion exchange columns and sephadex SephadexG-
25 are purchased from GE companies of the U.S.；McCoys 5A culture mediums, hyclone are purchased from Gibco companies of the U.S.；Pen .- Strep
Solution is purchased from Hyclone companies of the U.S.；CCK-8 kits are purchased from Japanese colleague's chemistry institute；It is pure that remaining reagent is analysis.
(4) key instrument：5804R high speed freezing centrifuges, German Eppendorf companies；HH-6 digital display waters bath with thermostatic control
Pot, Changzhou Guohua Electric Appliance Co., Ltd.；PB-10pH meters, electronic analytical balance, German Sartorius companies；Gl5 4-DWS are high
Pressure autoclave, Zealway companies of the U.S.；Class II Type B2 type Biohazard Safety Equipments, German ESCO companies；BPN-80CRH
CO2gas incubator, Kunshan Yiheng Scientific Instruments Co., Ltd；Infinite M200RQ ELIASAs, NanoQuant companies of Switzerland；
XDS-IA inverted biologic microscopes, Shanghai precision instrumentation Co., Ltd.
2nd, process of the test is prepared
(1) enzymolysis process flow：From pepsin, neutral proteinase, flavor protease, trypsase, basic protein
5 kinds of protease of enzyme are digested to Ruditapes philippinarum meat respectively, and the enzymatic hydrolysis condition of various protease is shown in Table 1, and enzymolysis process is successively
It is as follows：A certain amount of Ruditapes philippinarum homogenate is weighed, isometric distilled water (solid-liquid ratio 1 is added:1), 0.5mol/L NaOH
Or 0.5mol/L HCl solutions adjust pH value, enzyme-added to be hydrolyzed under the conditions of respective optimum temperature and pH, boiling water bath goes out enzyme 10min, from
The heart (8000r/min, 5min), takes supernatant, freeze-drying, the antitumor activity of CCK-8 method Preliminary Determination enzymolysis products.
The enzymatic hydrolysis condition of several protease of table 1
| Enzyme class | PH | Temperature/DEG C | Enzyme concentration/(U/g) | Enzymolysis time/h |
| Pepsin (Pepsin) | 2.0 | 37 | 1000 | 6 |
| Neutral proteinase Neutral | 7.0 | 50 | 1000 | 6 |
| The white enzyme Flavor of local flavor | 7.0 | 50 | 1000 | 6 |
| Trypsase Trypsin | 8.0 | 37 | 1000 | 6 |
| Alkali protease Alcalase | 10.0 | 50 | 1000 | 6 |
(2) cell culture：A549 non-small lung cancers cell is with containing 10% hyclone and 0.1% Pen .- Strep
McCoy ' s 5A cultures are based on 37 DEG C, are cultivated in 5%CO2 incubators to exponential phase.
(3) measure of cell proliferation inhibition rate：Detect medicine to A549 non-small lung cancers cell inhibitory effects using CCK-8 methods
Effect.From the A549 non-small lung cancers cells in exponential phase, 96 holes are inoculated according to 2.5 × 104cells/mL density
Plate, culture medium is abandoned after 24h is cultivated under the conditions of every μ L, 5%CO2,37 DEG C of hole 100, adds 100 μ L serum free mediums, cultivates 17h
After abandon culture medium.Control group (serum free medium+cell), dosing group (20mg/mL medicines+cell) and blank group (nothing are set
Blood serum medium, acellular), cultivate 24h under the conditions of every μ L, 5%CO2,37 DEG C of hole 100.Liquid in 96 orifice plates is abandoned, PBS is added
Cleaning twice, abandons PBS, adds 100 μ L CCK-8 solution (CCK-8 stostes：Serum free medium=1:10), 5%CO2,37 DEG C
Under the conditions of be incubated 3h, using ELIASA at 450nm mensuration absorbance (OD values), calculate medicine to A549 non-small lung cancers cell increase
Inhibiting rate (abbreviation A549 inhibiting rates) is grown, every group of experiment sets 6 parallel multiple holes.
B=(A1-A2)/(A1-A3) × 100
In formula：B-A549 inhibiting rates/%；The OD values of A1-control group；The OD values of A2-dosing group；A3-blank group
OD values.
(4) Ruditapes philippinarum natineoplaston initial gross separation：From the milipore filter that molecule interception is 5KDa and 10KDa to stomach
Protease hydrolyzed liquid is retained, and obtains MWCO I (molecular weight be more than 10KDa), (molecular weight is between 5KDa and 10KDa for MWCO II
Between) and three ultrafiltration components of MWCO III (molecular weight is less than 5KDa), and investigate raw material, MWCO I, MWCO II and MWCO III couple
The inhibited proliferation of A549 non-small lung cancers cells, primarily determines that the molecular weight ranges having compared with powerful antitumor activity polypeptide, greatly
Amount collects the Ruditapes philippinarum natineoplaston of the molecular weight ranges, freeze-drying.
(5) strong cat ion exchange column chromatographic isolation：High anti-tumor activity product in above-mentioned steps is freezed after dialysis
Dry, by the product after freeze-drying by SP Sephadex C-25 strong cat ion exchange columns, the second of 0~1mol/L NaCl
Sour sodium-acetate buffer linear elution, elution speed is 1mL/min, and ultraviolet-visible spectrophotometer 280nm is detected, point
Each eluting peak is not collected, and dried each component is carried out antitumor activity point by desalination of dialysing, freeze-drying using CCK-8 methods
Analysis, the eluting peak where further determining that target peptide, and the elution fraction is largely collected, dialyse desalination, freeze-drying.
(6) gel column chromatography is separated：Target peptide after freeze-drying in above-mentioned steps is crossed into Sephadex G-25 gels
Post, mobile phase is ultra-pure water, and 0.5~1.5mL/min of elution speed, ultraviolet-visible spectrophotometer 280nm is detected, point
Each eluting peak is not collected, and dried each component is carried out antitumor activity analysis, further by freeze-drying using CCK-8 methods
Eluting peak where determining target peptide, and the elution fraction is largely collected, freeze-drying.
(7) influences of the MWCO III to A549 tumor-bearing mices is investigated：1. A549 bearing mouse models are set up, is claimed daily before inoculation
Nude mouse body weight of amount, body weight to be averaged carries out modeling when reaching 20g or so, using nude mice by subcutaneous grafting.First, it is right to take
The A549 non-small lung cancers cells of number phase growth, after pancreatin digestion, single cell suspension are prepared into using serum free medium is resuspended, are adjusted
Whole cell number is 1 × 107cells/mL, and 1mL syringes draw 0.2mL A549 non-small lung cancers cell suspension inoculations in after mouse
Limb drosal part.2. it is grouped and is administered, after modeling five days, can Palpable and the skin to grain of rice size if these mouse hind leg drosal parts are subcutaneous
The success of lower tumour, as modeling.When tumor average diameter reaches 5mm, 5 groups are randomly divided into, every group 8, group is：Model group,
Basic, normal, high dosage group (MWCO III, 100,200,400mg/kg) and fluorouracil group (2mg/kg).Administering mode：Note in abdominal cavity
Penetrate, 0.2mL//d, continuous 16d.
The de- neck of mouse is put to death, knurl body is peeled off, assay balance is weighed, and records knurl weight, and calculate tumor suppression as follows
Rate/%.
Tumour inhibiting rate=(model group knurl weight-dosing group knurl weight)/model group knurl weight × 100%.
3rd, result
(1) optimum protein enzyme determines
As shown in figure 1, the enzymolysis product that 5 kinds of protease is obtained is respectively provided with suppression and makees to A549 non-small lung cancers cell propagation
With, wherein pepsin enzymolysis product is most strong to the proliferation inhibition activity of A549 non-small lung cancers cells, when concentration is 20mg/mL,
Effect 24h, inhibiting rate is 99.22%.As shown in Fig. 2 three components, originals that pepsin enzymolysis liquid is obtained through milipore filter retention
After material effect 24h, MWCO III is most strong to the proliferation inhibition activity of A549 non-small lung cancers cells, and its IC50 is 6.88mg/mL.
(2) Ruditapes philippinarum natineoplaston is isolated and purified
As shown in figure 3, MWCO III through SP Sephadex C-25 strong cations post elute after, be obtained 6 components (I~
Ⅵ).As shown in figure 4, the antitumor activity highest of peak IV, after A549 non-small lung cancers cytosiies 24h, its IC50 is 6.65mg/
mL.As shown in figure 5, peak IV obtains 3 components (A~C) after being eluted through Sephadex G-25 gel columns.As shown in fig. 6, peak A is anti-
Tumor promotion highest, after A549 non-small lung cancers cytosiies 24h, its IC50 is 4.82mg/mL.
(3) A549 tumor-bearing mices vivo test
As shown in fig. 7, the MWCO III of basic, normal, high dosage is respectively provided with tumor killing effect, the wherein tumor killing effects of high dose MWCO III
Most preferably, tumour inhibiting rate is 49.01%.High dose MWCO III is detected, it contains 15 kinds of amino acid, its Glutamic Acid, bright ammonia
The acidic amino acid content such as acid and aspartic acid is higher, and 25.43%, 9.16% and the 8.51% of its total amino acid is accounted for respectively, sees
Table 2.
Table 2MWCOIII amino acid composition analysises
| Amino acid | Content/% |
| Aspartic acid | 8.51 |
| Glutamic acid | 25.43 |
| Serine | 5.50 |
| Glycine | 5.39 |
| Arginine | 3.45 |
| Threonine | 4.74 |
| Alanine | 7.33 |
| Proline | 3.23 |
| Tyrosine | 3.99 |
| Valine | 5.28 |
| Methionine | 4.20 |
| Isoleucine | 3.77 |
| Leucine | 9.16 |
| Phenylalanine | 4.74 |
| Lysine | 5.28 |
This experiment chooses 5 kinds of protease and Ruditapes philippinarum meat is digested first, compares same concentration conditions and descends each egg
White enzyme enzymolysis product finds to be produced with different protease hydrolyzeds under concentration conditions to the proliferation inhibition activity of A549 non-small lung cancers cells
Thing is different to the proliferation inhibition activity of A549 non-small lung cancers cells, and this should be relevant with the Nomenclature Composition and Structure of Complexes of enzymolysis product.Accordingly
Speculate, when obtaining antitumor activity peptide using enzyme solution, the selectivity of enzyme is most important, under conditions of conditions permit, should
Several protease of multiselect carry out screening active ingredients as far as possible.Secondly, during acquisition target peptide is isolated and purified, different points are detected
The component of son amount and different eluting peaks, its antitumor activity also has very big difference, and in ultrafiltration retention component, molecular weight is less than
The component antitumor activity highest of 5KDa, and in polypeptide in the range of this, play mainly certain peak of active function
Polypeptide fractions, with the carrying out for isolating and purifying, the antitumor activity of polypeptide gradually strengthens, and its IC50 value is by 6.883mg/mL reductions
To 4.82mg/mL, it is seen then that be very important during high activity polypeptide is obtained isolating and purifying.Finally, MWCO has been investigated
III pair of influence of A549 tumor-bearing mices, result of the test shows that the MWCO III of high dose has very well in A549 tumor-bearing mice bodies
Tumor killing effect.
To sum up, the Ruditapes philippinarum natineoplaston high dose ultrafiltration component that the present invention is prepared is to A549 tumors
The inhibiting rate of body is high, and high activity polypeptide is obtained by separation purifying technique of the invention, with great application value.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, the change or replacement that can be readily occurred in,
Should all be included within the scope of the present invention.
Claims (6)
1. the preparation method of Ruditapes philippinarum high activity natineoplaston, it is characterised in that comprise the following steps：
S1. digest, take fresh Ruditapes philippinarum, after seawater tells sand, after being put into refrigerator freezing, open shell and take meat, be homogenized, solid-liquid ratio 1:
1, adjust pH value 2.0, add pepsin, enzyme concentration be 500~3000U/g, 31~43 DEG C of hydrolysis temperature, enzymolysis time 2~
12 h, are centrifuged after the enzyme that goes out, and take supernatant liquor；
S2. ultrafiltration, the supernatant liquor that step S1 is obtained carries out ultrafiltration retention, collects the trapped fluid of below molecular weight 5KDa, receives
Filter concentrating and desalinating, freeze-drying；
S3. strong cat ion exchange column chromatographic isolation, the product of freeze-drying is by strong cat ion exchange column, buffer solution in step S2
After linear elution, detected at ultraviolet-visible spectrophotometer 280nm, each eluting peak is collected respectively, desalination of dialysing is cold
It is lyophilized dry；Dried each component is carried out into antitumor activity analysis, the eluting peak where further determining that target peptide, and to this
Elution fraction is largely collected, freeze-drying；
S4. gel column chromatography is separated, and the product after freeze-drying in step S3 is crossed into gel column wash-out, then in ultraviolet-visible
Detected at spectrophotometer 280nm, each eluting peak is collected respectively, dialyse desalination, freeze-drying, by dried each component
Antitumor activity analysis is carried out, the eluting peak where further determining that target peptide, and the elution fraction is largely collected, freezing is dry
It is dry to obtain final product Ruditapes philippinarum high activity natineoplaston.
2. the preparation method of Ruditapes philippinarum high activity natineoplaston as claimed in claim 1, it is characterised in that in step S3
Using SP Sephadex C-25 strong cat ion exchange columns, buffer solution be containing concentration less than or equal to 1mol/L NaCl acetic acid receive-
Acetate buffer carries out linear elution, and elution speed is 0.5~1.5mL/min.
3. the preparation method of Ruditapes philippinarum high activity natineoplaston as claimed in claim 1 or 2, it is characterised in that step
Sephadex G-25 gel columns are used in S4, mobile phase is eluted for ultra-pure water, elution speed is 0.5~1.5 mL/min.
4. the preparation method of Ruditapes philippinarum high activity natineoplaston as claimed in claim 3, it is characterised in that step S3 and
Antitumor activity analysis uses CCK-8 methods in S4.
5. the preparation method of Ruditapes philippinarum high activity natineoplaston as claimed in claim 4, it is characterised in that in step S1
The enzyme concentration of pepsin is 1000U/g, and hydrolysis temperature is 37 DEG C, and enzymolysis time is 6h.
6. the Ruditapes philippinarum that the preparation method of the Ruditapes philippinarum high activity natineoplaston described in Claims 1 to 5 is obtained resists
Application of the tumour peptide on A549 non-small lung cancers are suppressed.
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| CN102372765A (en) * | 2010-08-05 | 2012-03-14 | 浙江海洋学院 | Ruditapes philippinarum enzymatic polypeptide and preparation method thereof |
| CN102558296A (en) * | 2010-12-16 | 2012-07-11 | 浙江海洋学院 | Mytilus edulis enzymolysis polypeptide and preparation method and application thereof |
| CN104212861A (en) * | 2013-05-29 | 2014-12-17 | 浙江海洋学院 | Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer |
| CN104337837A (en) * | 2013-07-23 | 2015-02-11 | 浙江海洋学院 | Anti-lung cancer application of ruditapes philippinarum enzymatic hydrolysate |
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2017
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