CN109182428A - Mulberry silkworm chrysalis anticancer active peptide BPP-2 and the preparation method and application thereof - Google Patents
Mulberry silkworm chrysalis anticancer active peptide BPP-2 and the preparation method and application thereof Download PDFInfo
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- CN109182428A CN109182428A CN201810954614.1A CN201810954614A CN109182428A CN 109182428 A CN109182428 A CN 109182428A CN 201810954614 A CN201810954614 A CN 201810954614A CN 109182428 A CN109182428 A CN 109182428A
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 97
- 101800000103 Bradykinin-potentiating peptide 10c Proteins 0.000 title claims abstract description 37
- 101800001668 Bradykinin-potentiating peptide-2 Proteins 0.000 title claims abstract description 37
- 230000001093 anti-cancer Effects 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 33
- 239000012498 ultrapure water Substances 0.000 claims abstract description 33
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 26
- 239000000843 powder Substances 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 239000012528 membrane Substances 0.000 claims abstract description 19
- 229920005654 Sephadex Polymers 0.000 claims abstract description 9
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 9
- 108091005804 Peptidases Proteins 0.000 claims abstract description 8
- 239000004365 Protease Substances 0.000 claims abstract description 8
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 8
- 108010056079 Subtilisins Proteins 0.000 claims abstract description 8
- 238000005238 degreasing Methods 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 238000000502 dialysis Methods 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 6
- 102000005158 Subtilisins Human genes 0.000 claims abstract description 5
- 239000003513 alkali Substances 0.000 claims abstract description 5
- 239000002253 acid Substances 0.000 claims abstract description 3
- 239000006228 supernatant Substances 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 230000002255 enzymatic effect Effects 0.000 claims description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 9
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 9
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 9
- 238000004090 dissolution Methods 0.000 claims description 7
- 238000009835 boiling Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000012466 permeate Substances 0.000 claims description 5
- 239000003208 petroleum Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 4
- 229940041181 antineoplastic drug Drugs 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 238000002835 absorbance Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 230000005540 biological transmission Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 17
- 230000012010 growth Effects 0.000 abstract description 11
- 206010067482 No adverse event Diseases 0.000 abstract description 7
- 210000003292 kidney cell Anatomy 0.000 abstract description 7
- 210000002540 macrophage Anatomy 0.000 abstract description 7
- 238000009777 vacuum freeze-drying Methods 0.000 abstract description 4
- 230000000118 anti-neoplastic effect Effects 0.000 abstract description 2
- 241000382353 Pupa Species 0.000 description 12
- 238000000034 method Methods 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 8
- 206010017758 gastric cancer Diseases 0.000 description 5
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 238000013019 agitation Methods 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- 235000008708 Morus alba Nutrition 0.000 description 3
- 240000000249 Morus alba Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
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- 238000003556 assay Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
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- 241000238631 Hexapoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000020939 nutritional additive Nutrition 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses a kind of mulberry silkworm chrysalis anticancer active peptide BPP-2 and the preparation method and application thereof.Mulberry silkworm chrysalis processing after fresh cocoon reeling is obtained into mulberry silkworm chrysalis powder, after taking mulberry silkworm chrysalis powder degreasing, is successively handled under alkalinity and acid condition, then bag filter dialysis, obtains mulberry silkworm chrysalis albumen.Mulberry silkworm chrysalis albumen alcalase alkali protease is taken to digest, enzymolysis liquid carries out ultra-filtration and separation, take molecular mass 5-10kDa ultrafiltrate, upper Sephadex G-50 gel after filter membrane, it is eluted with ultrapure water, the 2nd corresponding eluent of eluting peak is collected, obtains the product BPP-2 after vacuum freezedrying.Present invention preparation product purity is uniform, inhibited to the growth of SGC-7901 cells, has no adverse effects to normal cell human embryonic kidney cell HEK293 and mouse macrophage RAW264.7 growth, can be used for antineoplastic product exploitation.
Description
Technical field
The present invention relates to active peptide preparation fields, more particularly, to a kind of mulberry silkworm chrysalis anticancer active peptide BPP-2 and its system
Preparation Method and application.
Background technique
Natural activity peptide not only has the growth and development for promoting body and metabolism, anticancer, anti-oxidant, the immune function of raising
Can etc. important function, but also possess and easily digested and assimilated and the characteristics such as toxic side effect is small by body.For this purpose, from native protein
It extracts active peptide and is applied in the fields such as health food, Medicines, be the deep research paid high attention to by domestic and international researcher
Hot spot.
Mulberry silkworm chrysalis is the pupa of silkworm (Bombyx mori), has more than 1400 years edible and medicinal histories in China, be by
The Ministry of Public Health is included in unique insect based food in " the food resource list as ordinary food management ".Mulberry silkworm chrysalis is rich in albumen
Matter, protein content account for the 45-50% of silkworm chrysalis dry weight, and mostly high-quality protein.China is maximum silk in the world
Producing country produces about 650,000 tons of fresh cocoon per year, about 300,000 tons of silkworm chrysalis can be produced after filature, Silkworm Pupa Resources are very rich.But it is current very big
Directly as animal and fowl fodder and nutritional additive, some are also taken as fertilizer or directly discarded, cause resource unrestrained part mulberry silkworm chrysalis
Take.Separation prepares mulberry silkworm chrysalis anticancer active peptide, for promoting the medicinal exploitation of mulberry silkworm chrysalis and Silkworm Pupa Resources higher value application etc.
With positive realistic meaning.
Summary of the invention
In order to solve the problems in background technique, the present invention provides a kind of mulberry silkworm chrysalis anticancer active peptide BPP-2 and its systems
Preparation Method and application, using protease hydrolyzed, ultra-filtration and separation, the method for Sephadex G-50 gel-purified, have been prepared mulberry
Silkworm chrysalis anticancer active peptide BPP-2, mulberry silkworm chrysalis anticancer active peptide BPP-2 are that inventor's one kind of separation and Extraction from mulberry silkworm chrysalis is novel
Active peptide is simultaneously named as BPP-2.
The technical solution adopted by the invention is as follows:
One, a kind of preparation method of mulberry silkworm chrysalis anticancer active peptide BPP-2:
1) the mulberry silkworm chrysalis processing after fresh cocoon reeling is obtained into mulberry silkworm chrysalis powder；
2) successively processing obtains mulberry silkworm chrysalis albumen under alkalinity and acid condition after the degreasing of mulberry silkworm chrysalis powder；
3) mulberry silkworm chrysalis albumen is digested with alcalase alkali protease, is then centrifuged for handling and collecting supernatant, on
It is re-dissolved in ultrapure water after clear liquid freeze-drying and obtains mulberry silkworm chrysalis protein enzymatic hydrolyzate；
4) mulberry silkworm chrysalis protein enzymatic hydrolyzate carries out ultra-filtration and separation, collects molecular mass 5-10kDa ultrafiltrate；
5) upper Sephadex G-50 gel column carries out separation elution after ultrafiltrate filter membrane, collects washing for the 2nd eluting peak
De- liquid, obtains uniform mulberry silkworm chrysalis activity peptide composition BPP-2 after freeze-drying.
The concrete technology condition of the preparation method is as follows:
Step 1): the mulberry silkworm chrysalis after fresh cocoon reeling is dried in vacuo 36h at -50 DEG C, pulverizes 5min at -15 DEG C,
Obtain mulberry silkworm chrysalis powder；
Step 2): taking mulberry silkworm chrysalis powder to be added in petroleum ether solution, repeats soak degreasing 3 times at 25 DEG C, when each degreasing
Between for for 24 hours, filter, filter residue, which dries in 40 DEG C and crosses 80-100 mesh, obtains filter residue powder, will filter residue powder ultrapure water is added after carry out
Magnetic agitation sufficiently dissolves, and with 1M NaOH tune pH to 9.5, stirs 1h, collects after 10min is centrifuged under the revolving speed of 5000rpm
Then clear liquid adjusts supernatant pH to 4 with 1M HCl, 10min is centrifuged under the revolving speed of 5000rpm after standing 30min, and it is heavy to collect
It forms sediment, after precipitating is dissolved with ultrapure water, with 14000Da bag filter dialysis 48h, obtains mulberry silkworm chrysalis albumen；
Step 3): mulberry silkworm chrysalis albumen is digested with alcalase alkali protease, after enzymatic hydrolysis, is immediately placed in boiling water
15min enzymolysis reaction, is cooled to room temperature, and collects supernatant after being centrifuged 10min under the revolving speed of 5000rpm, supernatant-
It is dissolved in ultrapure water after being dried in vacuo at 50 DEG C, obtains the mulberry silkworm chrysalis protease that quality percent by volume is 0.6% (g/mL)
Solve liquid；
Step 4): it uses trapped molecular weight to carry out ultrafiltration to mulberry silkworm chrysalis protein enzymatic hydrolyzate for the ultrafiltration membrane of 10kDa, takes
Liquid is crossed, then the ultrafiltration membrane for being 5kDa with trapped molecular weight carries out ultrafiltration to permeate, takes trapped fluid to get molecular mass 5- is arrived
The component of 10kDa, being completely dissolved in after being dried in vacuo at -50 DEG C and being configured to mass concentration in ultrapure water is the molten of 3mg/mL
Liquid, 12000rpm are centrifuged 10min, take supernatant ultrafiltrate；
Step 5): supernatant ultrafiltrate obtained by step 4) is crossed into 0.22 μm of filter membrane, upper Sephadex G-50 gel column, loading
Amount is 1mL, is eluted with ultrapure water, and elution flow rate 0.5-0.7mL/min is collected with automatic collector, utilizes light splitting light
Degree is counted detects every pipe absorbance value at 280nm, drafting elution curve, the eluent of the 2nd eluting peak of collection, true at -50 DEG C
Vacuum freecing-dry obtains uniform mulberry silkworm chrysalis activity peptide composition, is named as BPP-2.
The mass volume ratio (W:V) of mulberry silkworm chrysalis powder and petroleum ether solution is 1:3, filter residue powder and ultrapure water in the step 2)
Mass volume ratio (W:V) be 1:10.
The enzymatic hydrolysis condition of the step 3) are as follows: the mass concentration of mulberry silkworm chrysalis albumen is 10mg/mL, enzymolysis time be 160~
180min, hydrolysis temperature are 60 DEG C, enzymatic hydrolysis pH is 9.0, enzyme concentration is 3500U/g mulberry silkworm chrysalis albumen quality.
The ultra-filtration conditions of the step 4) are as follows: 3L mulberry silkworm chrysalis protein enzymatic hydrolyzate, 1.30MPa are into film pressure, 0.30MPa membrane
Pressure and 25 DEG C of operating temperatures.
Two, a kind of application of mulberry silkworm chrysalis anticancer active peptide BPP-2
The mulberry silkworm chrysalis anticancer active peptide BPP-2 is used to prepare anticancer drug.
The anticancer drug is for resisting SGC-7901 cells.
The beneficial effects of the present invention are:
The present invention isolates and purifies the mulberry silkworm chrysalis anticancer active peptide BPP-2 of preparation, and purity is uniform, to gastric carcinoma cells SGC-
7901 growth shows apparent inhibiting effect, to normal cell human embryonic kidney cell HEK293 and mouse macrophage
RAW264.7 growth has no adverse effects, and can be used for the exploitation of antineoplastic product, this has product for promoting mulberry silkworm chrysalis medical value
The Social benefit and economic benefit of pole.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Before implementation, the mulberry silkworm chrysalis after fresh cocoon reeling is first dried in vacuo 36h at -50 DEG C, is pulverized at -15 DEG C
5min obtains mulberry silkworm chrysalis powder；Mulberry silkworm chrysalis powder is taken, petroleum ether solution is added by 1:3 mass volume ratio (W:V), after mixing, at 25 DEG C
Lower soak degreasing 3 times carries out suction filtration processing with suction filtration machine, takes filter residue every time for 24 hours, dries in 40 DEG C.Filter residue is dried for following
Each embodiment.
The embodiment of the present invention, to the growing state of SGC-7901 cells, is used using mtt assay detection BPP-2
Lowry method surveys protein content, prepares the technical effect of product in specific implementation for verifying the present invention.
Embodiment 1:
Drying filter residue is taken, 80 meshes are crossed, ultrapure water is added by 1:10 mass volume ratio (W:V) in filter residue powder, and magnetic agitation is filled
After dividing dissolution, with 1M NaOH tune pH=9.5,5000rpm is centrifuged 10min after stirring 1h, collects supernatant, supernatant 1M
HCl adjusts pH=4, and 5000rpm is centrifuged 10min after standing 30min, collects precipitating and uses after precipitating a small amount of ultrapure water dissolution
14000Da bag filter dialysis 48h, obtains silkworm pupa protein liquid.
It is with the protein concentration of Lowry method measurement silkworm pupa protein liquid and with ultrapure water and 1M NaOH tune mass concentration
10mg/mL and pH=9.0, adding alcalase basic protein enzyme amount is 3500U/g mulberry silkworm chrysalis albumen quality, is digested at 60 DEG C
160min after enzymatic hydrolysis, is immediately placed in 15min enzymolysis reaction in boiling water, is cooled to room temperature, 5000rpm centrifugation
10min is dried in vacuo at -50 DEG C of supernatant, is re-dissolved in ultrapure water, obtains the mulberry that mass percent is 0.6% (g/mL)
Pupa albumen enzymolysis liquid.
Into film pressure 1.30MPa, go out film pressure 0.30MPa, under 25 DEG C of operating temperature of operating condition, first with retention point
The ultrafiltration membrane that protonatomic mass is 10kDa carries out ultrafiltration to 0.6% mulberry silkworm chrysalis protein enzymatic hydrolyzate, takes permeate, then with retaining molecule matter
The ultrafiltration membrane that amount is 5kDa carries out ultrafiltration, takes trapped fluid, obtains molecular mass 5-10kDa component, be dried in vacuo at -50 DEG C, then
It is that 3mg/mL is completely dissolved in ultrapure water by mass concentration, 12000rpm is centrifuged 10min, takes supernatant ultrafiltrate；
Supernatant ultrafiltrate crosses 0.22 μm of filter membrane, upper Sephadex G-50 gel column, with ultrapure water in 0.7mL/min flow velocity
Elution, automatic collector is collected, and with spectrophotometry, collects the eluent with the 2nd eluting peak of protein, and -50
Vacuum freeze drying at DEG C obtains mulberry silkworm chrysalis active peptide BPP-2.
Result of implementation: BPP-2 purity is uniform (protein content 99.40%), in concentration 1.28mg/mL, to human gastric cancer
The inhibiting rate of cell SGC-7901 is 62.51%, to normal cell human embryonic kidney cell HEK293 and mouse macrophage
RAW264.7 growth has no adverse effects.
Embodiment 2:
Drying filter residue is taken, 90 meshes are crossed, ultrapure water is added by 1:10 mass volume ratio (W:V) in filter residue powder, and magnetic agitation is filled
After dividing dissolution, with 1M NaOH tune pH=9.5,5000rpm is centrifuged 10min after stirring 1h, collects supernatant, supernatant 1M
HCl adjusts pH=4, and 5000rpm is centrifuged 10min after standing 30min, collects precipitating and uses after precipitating a small amount of ultrapure water dissolution
14000Da bag filter dialysis 48h, obtains silkworm pupa protein liquid.
It is with the protein concentration of Lowry method measurement silkworm pupa protein liquid and with ultrapure water and 1M NaOH tune mass concentration
10mg/mL and pH=9.0, adding alcalase basic protein enzyme amount is 3500U/g mulberry silkworm chrysalis albumen quality, is digested at 60 DEG C
170min after enzymatic hydrolysis, is immediately placed in 15min enzymolysis reaction in boiling water, is cooled to room temperature, 5000rpm centrifugation
10min is dried in vacuo at -50 DEG C of supernatant, is re-dissolved in ultrapure water, obtains the mulberry that mass percent is 0.6% (g/mL)
Pupa albumen enzymolysis liquid.
Into film pressure 1.30MPa, go out film pressure 0.30MPa, under 25 DEG C of operating temperature of operating condition, first with retention point
The ultrafiltration membrane that protonatomic mass is 10kDa carries out ultrafiltration to 0.6% mulberry silkworm chrysalis protein enzymatic hydrolyzate, takes permeate, then with retaining molecule matter
The ultrafiltration membrane that amount is 5kDa carries out ultrafiltration, takes trapped fluid, obtains molecular mass 5-10kDa component, be dried in vacuo at -50 DEG C, then
It is that 3mg/mL is completely dissolved in ultrapure water by mass concentration, 12000rpm is centrifuged 10min, takes supernatant ultrafiltrate；
Supernatant ultrafiltrate crosses 0.22 μm of filter membrane, upper Sephadex G-50 gel column, with ultrapure water in 0.6mL/min flow velocity
Elution, automatic collector is collected, and with spectrophotometry, collects the eluent with the 2nd eluting peak of protein, and -50
Vacuum freeze drying at DEG C obtains mulberry silkworm chrysalis active peptide BPP-2.
Result of implementation: BPP-2 purity is uniform (protein content 99.48%), in concentration 1.28mg/mL, to human gastric cancer
The inhibiting rate of cell SGC-7901 is 62.95%, to normal cell human embryonic kidney cell HEK293 and mouse macrophage
RAW264.7 growth has no adverse effects.
Embodiment 3:
Drying filter residue is taken, is sieved with 100 mesh sieve, ultrapure water is added by 1:10 mass volume ratio (W:V) in filter residue powder, and magnetic agitation is filled
After dividing dissolution, with 1M NaOH tune pH=9.5,5000rpm is centrifuged 10min after stirring 1h, collects supernatant, supernatant 1M
HCl adjusts pH=4, and 5000rpm is centrifuged 10min after standing 30min, collects precipitating and uses after precipitating a small amount of ultrapure water dissolution
14000Da bag filter dialysis 48h, obtains silkworm pupa protein liquid.
It is with the protein concentration of Lowry method measurement silkworm pupa protein liquid and with ultrapure water and 1M NaOH tune mass concentration
10mg/mL and pH=9.0, adding alcalase basic protein enzyme amount is 3500U/g mulberry silkworm chrysalis albumen quality, is digested at 60 DEG C
180min after enzymatic hydrolysis, is immediately placed in 15min enzymolysis reaction in boiling water, is cooled to room temperature, 5000rpm centrifugation
10min is dried in vacuo at -50 DEG C of supernatant, is re-dissolved in ultrapure water, obtains the mulberry that mass percent is 0.6% (g/mL)
Pupa albumen enzymolysis liquid.
Into film pressure 1.30MPa, go out film pressure 0.30MPa, under 25 DEG C of operating temperature of operating condition, first with retention point
The ultrafiltration membrane that protonatomic mass is 10kDa carries out ultrafiltration to 0.6% mulberry silkworm chrysalis protein enzymatic hydrolyzate, takes permeate, then with retaining molecule matter
The ultrafiltration membrane that amount is 5kDa carries out ultrafiltration, takes trapped fluid, obtains molecular mass 5-10kDa component, be dried in vacuo at -50 DEG C, then
It is that 3mg/mL is completely dissolved in ultrapure water by mass concentration, 12000rpm is centrifuged 10min, takes supernatant ultrafiltrate；
Supernatant ultrafiltrate crosses 0.22 μm of filter membrane, upper Sephadex G-50 gel column, with ultrapure water in 0.5mL/min flow velocity
Elution, automatic collector is collected, and with spectrophotometry, collects the eluent with the 2nd eluting peak of protein, and -50
Vacuum freeze drying at DEG C obtains mulberry silkworm chrysalis active peptide BPP-2.
Result of implementation: BPP-2 purity is uniform (protein content 99.51%), in concentration 1.28mg/mL, to human gastric cancer
The inhibiting rate of cell SGC-7901 is 63.15%, to normal cell human embryonic kidney cell HEK293 and mouse macrophage
RAW264.7 growth has no adverse effects.
The multiple implementation being not limited to the above embodiments according to the method for the present invention, all experiments obtain average data:
The mulberry silkworm chrysalis activity peptide composition BPP-2 being repeatedly prepared uses Lowry method to measure protein content as 99.42%, uses
Mtt assay detection BPP-2 shows apparent inhibiting effect to the growth of SGC-7901 cells, in concentration 1.28mg/mL
When inhibiting rate 62.89% (as shown in table 1), to normal cell human embryonic kidney cell HEK293 and mouse macrophage
RAW264.7 growth has no adverse effects, and is respectively 105.62% relative to cellular control unit vigor in concentration 1.28mg/mL
With 104.58% (as shown in table 2).
The inhibiting rate that 1 various concentration BPP-2 of table grows SGC-7901 cells
2 normal cell of table is at the BPP-2 of various concentration relative to the cell viability of control group
It can be seen that the present invention isolates and purifies the mulberry silkworm chrysalis anticancer active peptide BPP-2 of preparation, purity is uniform, to human gastric cancer
The growth of cell SGC-7901 shows apparent inhibiting effect, to normal cell human embryonic kidney cell HEK293 and mouse macrophage
Cell RAW264.7 growth has no adverse effects, and has prominent significant technical effect.
Claims (7)
1. a kind of mulberry silkworm chrysalis anticancer active peptide BPP-2 and the preparation method and application thereof, it is characterised in that:
1) the mulberry silkworm chrysalis processing after fresh cocoon reeling is obtained into mulberry silkworm chrysalis powder；
2) successively processing obtains mulberry silkworm chrysalis albumen under alkalinity and acid condition after the degreasing of mulberry silkworm chrysalis powder；
3) mulberry silkworm chrysalis albumen is digested with alcalase alkali protease, is then centrifuged for handling and collecting supernatant, supernatant
It is re-dissolved in after freeze-drying in ultrapure water and obtains mulberry silkworm chrysalis protein enzymatic hydrolyzate；
4) mulberry silkworm chrysalis protein enzymatic hydrolyzate carries out ultra-filtration and separation, collects molecular mass 5-10kDa ultrafiltrate；
5) upper Sephadex G-50 gel column carries out separation elution after ultrafiltrate filter membrane, collects the elution of the 2nd eluting peak
Liquid obtains uniform mulberry silkworm chrysalis activity peptide composition BPP-2 after freeze-drying.
2. a kind of mulberry silkworm chrysalis anticancer active peptide BPP-2 according to claim 1 and the preparation method and application thereof, feature exists
In the concrete technology condition of the preparation method is as follows:
Step 1): the mulberry silkworm chrysalis after fresh cocoon reeling is dried in vacuo 36h at -50 DEG C, pulverizes 5min at -15 DEG C, obtains
Mulberry silkworm chrysalis powder；
Step 2): taking mulberry silkworm chrysalis powder to be added in petroleum ether solution, repeats soak degreasing 3 times at 25 DEG C, and each degreasing time is
For 24 hours, it filters, filter residue, which dries in 40 DEG C and crosses 80-100 mesh, obtains filter residue powder, carries out magnetic force after ultrapure water is added in filter residue powder
Stirring sufficiently dissolution is stirred and is centrifuged 10min in 5000rpm after 1h, collected supernatant, then used with 1M NaOH tune pH to 9.5
It is centrifuged 10min in 5000rpm after 1M HCl adjusting supernatant pH to 4, standing 30min, collects precipitating, precipitating is dissolved with ultrapure water
Afterwards, with 14000Da bag filter dialysis 48h, mulberry silkworm chrysalis albumen is obtained；
Step 3): mulberry silkworm chrysalis albumen is digested with alcalase alkali protease, after enzymatic hydrolysis, is immediately placed in boiling water
15min enzymolysis reaction, is cooled to room temperature, and collects supernatant after being centrifuged 10min under the revolving speed of 5000rpm, supernatant-
It is dissolved in ultrapure water after being dried in vacuo at 50 DEG C, obtains the mulberry silkworm chrysalis protease that quality percent by volume is 0.6% (g/mL)
Solve liquid；
Step 4): it uses trapped molecular weight to carry out ultrafiltration to mulberry silkworm chrysalis protein enzymatic hydrolyzate for the ultrafiltration membrane of 10kDa, takes transmission
Liquid, then the ultrafiltration membrane for being 5kDa with trapped molecular weight carry out ultrafiltration to permeate, take trapped fluid to get molecular mass 5- is arrived
The component of 10kDa, being completely dissolved in after being dried in vacuo at -50 DEG C and being configured to mass concentration in ultrapure water is the molten of 3mg/mL
Liquid, 12000rpm are centrifuged 10min, take supernatant ultrafiltrate；
Step 5): supernatant ultrafiltrate obtained by step 4) is crossed into 0.22 μm of filter membrane, upper Sephadex G-50 gel column, applied sample amount is
1mL is eluted with ultrapure water, and elution flow rate 0.5-0.7mL/min is collected with automatic collector, utilizes spectrophotometer
Every pipe absorbance value is detected at 280nm, draws elution curve, collects the eluent of the 2nd eluting peak, and vacuum is cold at -50 DEG C
It is lyophilized dry, obtains uniform mulberry silkworm chrysalis activity peptide composition, be named as BPP-2.
3. the preparation method of mulberry silkworm chrysalis anticancer active peptide BPP-2 according to claim 2, which is characterized in that the step
2) mass volume ratio of mulberry silkworm chrysalis powder and petroleum ether solution is 1:3 in, and the mass volume ratio of filter residue powder and ultrapure water is 1:10.
4. the preparation method of mulberry silkworm chrysalis anticancer active peptide BPP-2 according to claim 2, which is characterized in that the step
3) enzymatic hydrolysis condition are as follows: the mass concentration of mulberry silkworm chrysalis albumen is 10mg/mL, enzymolysis time is 160~180min, hydrolysis temperature is
60 DEG C, enzymatic hydrolysis pH be 9.0, enzyme concentration is 3500U/g mulberry silkworm chrysalis albumen quality.
5. the preparation method of mulberry silkworm chrysalis anticancer active peptide BPP-2 according to claim 2, which is characterized in that the step
4) ultra-filtration conditions are as follows: 3L mulberry silkworm chrysalis protein enzymatic hydrolyzate, 1.30MPa go out film pressure and 25 DEG C of work into film pressure, 0.30MPa
Temperature.
6. a kind of application of mulberry silkworm chrysalis anticancer active peptide BPP-2, it is characterised in that: the mulberry silkworm chrysalis anticancer active peptide BPP-2 is used
In preparing anticancer drug.
7. the application of mulberry silkworm chrysalis anticancer active peptide BPP-2 according to claim 6, it is characterised in that: the anticancer drug
For resisting SGC-7901 cells.
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