CN108359702A - The preparation method and application of Yi Zhong angler fracture tumour decapeptides - Google Patents
The preparation method and application of Yi Zhong angler fracture tumour decapeptides Download PDFInfo
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- CN108359702A CN108359702A CN201810040290.0A CN201810040290A CN108359702A CN 108359702 A CN108359702 A CN 108359702A CN 201810040290 A CN201810040290 A CN 201810040290A CN 108359702 A CN108359702 A CN 108359702A
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- angler
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The preparation method and application of Yi Zhong angler fracture tumour decapeptides, the preparation method are：a）Particle：Angler bone carried out with high-speed tissue mashing machine to smash processing to pieces；b）Enzymolysis：Neutral proteinase Dui angler bone meal is selected to digest, Cheng anglers bone meal enzymolysis liquid processed；c）Ultrafiltration：Ultrafiltration system is added in angler bone meal enzymolysis liquid, ultrafiltration is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD, is collected>10KD, 10 5KD, 5 3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried.
Description
Technical field
Of the present invention is the preparation method and application of Yi Zhong angler fracture tumour decapeptides, especially a kind of to forefront
The preparation method and application of the inhibited anglers fracture tumour decapeptide of adenocarcinoma cell.
Background technology
Traditional treatment means include mainly prostate cancer radical excision, hormone therapy, chemotherapy, radiotherapy etc., but
Cancer is difficult to eradicate after treatment, and recurrence rate is high.Therefore, efficient one kind, low toxicity are found from ocean and with relatively strong specific
New type antineoplastic medicine have important research significance and clinical meaning.
During processing of aquatic products, a large amount of leftover bits and pieces will produce（Including fish head, fish-skin, fin, fish tail, fish-bone and
It remains the flesh of fish）, quality accounts for about the 30%~55% of raw material fish, and the content of different parts collagen is differed from 20%~50%.
Angler（Lophius litulon）Belong to cold warm nature demersal fishes, volt seabed of often dwelling is distributed in North Western Pacific, China's production
In northern East China Sea, the Huanghai Sea and the Bohai Sea.Wo Guo anglers demand for exports in recent years is vigorous, and , anglers are significantly increased in yield and price
Exporting oneself becomes the emerging fishery in China.Ran and in the processing of , anglers, a large amount of fish-skins are discarded as leftover bits and pieces, this
Environment is not only polluted, the waste of resource is also resulted in.Currently, for the economic value added of Ti Gao angler skins, has scholar
Begin one's study extraction dermatan sulfate, chondroitin sulfate, collagen etc. in Cong angler processing byproducts.
Active peptide is the compound being connected with peptide bond by two or more amino acid, and important physiology is played in human body
Effect plays physiological function, such as：Adjust internal moisture, electrolyte balance；Promote wound healing；Repair cell improves cell
Metabolism, can play protective effect on cancer risk etc..Active peptide is generally obtained by digesting bioactive materials, and research finds that food proteins pass through
Protease hydrolyzed mode can obtain active peptides vdiverse in function.
Invention content
It is an object of the invention to overcome the shortcomings of the prior art, and one kind is provided and passes through the white Mei Dui angler fishes of Dan
Bone is digested to obtain a kind of polypeptide yield Gao angler fractures inhibited to prostate gland cancer cell, extraction
The preparation method and application of tumour decapeptide.
The purpose of the present invention is by following technical solution to complete, the preparation sides of Yi Zhong angler fracture tumour decapeptides
Method, its Xuan Yong angler bone as raw material, and the preparation method includes the following steps：
a）Particle：Angler bone carried out with high-speed tissue mashing machine to smash processing to pieces, powdery and microparticulate anglers is made
Bone meal；
b）Enzymolysis：Neutral proteinase Dui angler bone meal is selected to digest, Cheng anglers bone meal enzymolysis liquid processed；
c）Ultrafiltration：Ultrafiltration system is added in angler bone meal enzymolysis liquid, ultrafiltration is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD, is collected
>10KD, 10-5KD, 5-3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried,
d）Angler enzymolysis liquid is crossed into G-25 eluents and obtains 5 peaks, is screened through anticancer experiment in vitro, peak 2 and peak 3 have most
High antitumor activity, collect peak 2 and peak 3, freeze-drying further isolates and purifies and with efficient liquid phase purity detecting；
e）By reversed-phase high performance liquid chromatography (RT- HPLC) purifying and amino acid sequence detection：Peak 2 is detected with HPLC and is found, should
Molecule section only has 1 eluting peak, and carries out amino acid sequence detection, and amino acid sequence is Ala-Pro-Phe-His-Asp-
Ser-Asp-Gln-Asn- Cys；Make into ten peptide product of angler fracture tumour.
As preferred：The step b）Enzymatic hydrolysis condition is：pH:7.0-7.5, temperature：40-45 DEG C, solid-liquid ratio：1:1, when
Between：5-7h, enzyme concentration:1200-1500 u/g；In the step c, ten peptide product of angler fracture tumour made, ammonia
Base acid sequence is Ala-Pro-Phe-His-Asp-Ser-Asp-Gln-Asn- Cys.
The step d）In, the G-25 gels separation：By step c）The enzymolysis liquid of acquisition is dissolved with distilled water,
Centrifugation takes supernatant, crosses 0.45 μm of miillpore filter, sample solution 1.5ml is taken to cross the SephadexG-25 columns (cm of 90cm × 115
(ID)) it is, eluent with distilled water, balance and elution；Often pipe collects 3ml, is detected at λ 280nm, collects each peak eluent；
The step e）In, the reversed-phase high performance liquid chromatography (RT- HPLC)：Purified with RT-HPLC, chromatostrip
Part:1) sample pre-treatments:Sample is centrifuged with the water dissolution of 0.06%TFA to 0.6ml（12000-14000RPM centrifuges 10 points
Clock）Take supernatant；2) system: Agilent 1260 HPLC；3) pillar:ZorbaxSB-C18 4.6X250 5um；4) sample introduction body
Product:80-100ul；5) buffer solution, equilibration buffer:0.06%TFA elution buffers:The acetonitrile of 0.05%TFA；6) gradient:
0% -0 % B are eluted 4 minutes, and 0% -8% B is eluted 25 minutes, and 8%-100%B is eluted 1 minute, and 100% -100%B is eluted 5 minutes；7)
Flow velocity:1.0 ml/min；8）Detection:280nm/214nm.
A kind of application of the Suo Shu anglers fracture tumour decapeptide on, Suo Shu angler fracture tumour decapeptides are as function
Food is used for the additional functionality food of patients with prostate cancer, apparent to the inhibiting effect of prostate cancer.
The present invention digests angler fish-bone powder raw material by the white enzyme of egg a kind of to prostate gland cancer cell to obtain
Inhibited ten peptide product of angler fracture tumour has the characteristics that the polypeptide yield of extraction is high.
Description of the drawings
Fig. 1 is anticancer experiment in vitro screening figure of this invention Suo Shu anglers enzymolysis liquids after 25 eluents of G-.
Fig. 2 is that the molecule section obtained with HPLC detections of the present invention elutes peak value figure.
Fig. 3 is decapeptide of the present invention to the active influence diagram of 3 cell proliferations of PC-.
Specific implementation mode
Below in conjunction with specific embodiment and testing inspection figure etc., the present invention will be described in detail：Of the present invention one
The preparation method of Zhong angler fracture tumour decapeptides, its Xuan Yong angler bone as raw material, and the preparation method includes as follows
Step：
a）Particle：Angler bone carried out with high-speed tissue mashing machine to smash processing to pieces, powdery and microparticulate anglers is made
Bone meal；
b）Enzymolysis：Neutral proteinase Dui angler bone meal is selected to digest, Cheng anglers bone meal enzymolysis liquid processed；
c）Ultrafiltration：Ultrafiltration system is added in angler bone meal enzymolysis liquid, ultrafiltration is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD, is collected
>10KD, 10-5KD, 5-3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried,
d）Angler enzymolysis liquid is crossed into G-25 eluents and obtains 5 peaks, is screened through anticancer experiment in vitro, peak 2 and peak 3 have most
High antitumor activity, collect peak 2 and peak 3, freeze-drying further isolates and purifies and with efficient liquid phase purity detecting；
e）By reversed-phase high performance liquid chromatography (RT- HPLC) purifying and amino acid sequence detection：Peak 2 is detected with HPLC and is found, should
Molecule section only has 1 eluting peak, and carries out amino acid sequence detection, and amino acid sequence is Ala-Pro-Phe-His-Asp-
Ser-Asp-Gln-Asn- Cys；Make into ten peptide product of angler fracture tumour.
Step b of the present invention）In, enzymatic hydrolysis condition is：pH:7.0-7.5, temperature：40-45 DEG C, solid-liquid ratio：1:1, when
Between：5-7h, enzyme concentration:1200-1500 u/g；In the step c, ten peptide product of angler fracture tumour made, ammonia
Base acid sequence is Ala-Pro-Phe-His-Asp-Ser-Asp-Gln-Asn- Cys.
The step d）In, the G-25 gels separation：By step c）The enzymolysis liquid of acquisition is dissolved with distilled water,
Centrifugation takes supernatant, crosses 0.45 μm of miillpore filter, sample solution 1.5ml is taken to cross the SephadexG-25 columns (cm of 90cm × 115
(ID)) it is, eluent with distilled water, balance and elution；Often pipe collects 3ml, is detected at λ 280nm, collects each peak eluent；
The step e）In, the reversed-phase high performance liquid chromatography (RT- HPLC)：Purified with RT-HPLC, chromatostrip
Part:1) sample pre-treatments:Sample is centrifuged with the water dissolution of 0.06%TFA to 0.6ml（12000-14000RPM centrifuges 10 points
Clock）Take supernatant；2) system: Agilent 1260 HPLC；3) pillar:ZorbaxSB-C18 4.6X250 5um；4) sample introduction body
Product:80-100ul；5) buffer solution, equilibration buffer:0.06%TFA elution buffers:The acetonitrile of 0.05%TFA；6) gradient:
0% -0 % B are eluted 4 minutes, and 0% -8% B is eluted 25 minutes, and 8%-100%B is eluted 1 minute, and 100% -100%B is eluted 5 minutes；7)
Flow velocity:1.0 ml/min；8）Detection:280nm/214nm.
A kind of application of the Suo Shu anglers fracture tumour decapeptide on, Suo Shu angler fracture tumour decapeptides are as function
Food is used for the additional functionality food of patients with prostate cancer, apparent to the inhibiting effect of prostate cancer.
Experimental method：
One, the optimization of enzymatic hydrolysis condition：
1, neutral proteinase is selected, is digested with its optimum enzymolysis condition.Its enzymatic hydrolysis condition is：pH:7.0-7.5, temperature：
40-45 DEG C, solid-liquid ratio：1:1, the time：5-7h, enzyme concentration:1200-1500 u/g.
2, cell culture：Choose Human Prostate Cancer Cells PC-3 hormone independent cells（Original is thin purchased from Chinese Academy of Sciences Shanghai
Born of the same parents library, is preserved by this laboratory passage）.PC-3 uses the F12 cultures containing 10% fetal calf serum to be based on 37 DEG C, in 5% CO2 incubators
It cultivates to exponential phase.
3, ultrafiltration：Ultrafiltration system is added in sample, ultrafiltration is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD, is collected>10KD、
10-5KD, 5-3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried.And carry out active anticancer screening.Strongest point of activity
Son amount further purifies.
4, G-25 gels detach：Sample is dissolved with distilled water, is centrifuged, supernatant is taken, crosses 0.45 μm of miillpore filter,
Take sample solution 1.5ml to cross Sephadex G-25 columns (cm of 90 cm × 115 (ID)), be eluent with distilled water, balance and
Elution.Often pipe collects 3ml, is detected at 280 nm of λ, collects each peak eluent.
5, reversed-phase high performance liquid chromatography (RT-HPLC)：RT-HPLC is purified.Chromatographic condition:1) sample pre-treatments:It will
Sample, to 0.6ml, is centrifuged with the water dissolution of 0.06%TFA（12000-14000 RPM are centrifuged 10 minutes）Take supernatant；2) system:
Agilent 1260 HPLC；3) pillar: Zorbax SB-C18 4.6 X 250 5 um；4) sampling volume:80-100 ul；
5) buffer solution equilibration buffer:0.06%TFA elution buffers:The acetonitrile of 0.05%TFA；6) gradient: 0% – 0 % B
Elution 4 minutes, 0% -8% B are eluted 25 minutes, and 8% -100%B is eluted 1 minute, and 100% -100%B is eluted 5 minutes；7) it flows
Speed: 1.0 ml/min；8）Detection: 280 nm /214nm
Two experimental results：
1, ultrafiltration and antitumor activity screening：Ultrafiltration system is added in enzymolysis sample, is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD
Ultrafiltration is collected>10KD, 8-5KD, 5-3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried.PC-3 is carried out with MTT methods
Cell proliferation inhibition rate is tested, and is found<The enzymolysis liquid energy of 3KD molecular weight effectively inhibits PC-3 to be proliferated.Therefore it will<3KD molecular weight
Enzymolysis liquid carries out isolating and purifying for next step.
2, G-25 isolates and purifies result：, angler enzymolysis liquids as shown in Figure 1 cross G-25 eluents and obtain 5 peaks, through external
Anti-tumor experiment screens, and peak 2 and peak 3 have highest antitumor activity, collects peak 2 and peak 3, and freeze-drying further divides
From purifying and with efficient liquid phase purity detecting.
3, reversed-phase high performance liquid chromatography (RT- HPLC) purifying and amino acid sequence detection：Shown in Fig. 2, HPLC is used at peak 2
Detection finds that the molecule section only has 1 eluting peak, and carries out amino acid sequence detection, and amino acid sequence is Ala-Pro-
Phe-His-Asp-Ser-Asp-Gln-Asn- Cys。
4, influence of the decapeptide to PC-3 cell-proliferation activities：Using F12 as control group, compare various concentration decapeptide to PC-3
The influence of cell Proliferation.Each concentration, each time point PC-3 cell-proliferation activities as shown in Figure 3, indicate in Fig. 3：Decapeptide can be effective
Inhibit PC-3 proliferation, and is in concentration and time dependence.
Claims (3)
1. the preparation method of Yi Zhong angler fracture tumour decapeptides, its Xuan Yongs angler bone as raw material, it is characterised in that described
Preparation method include the following steps：
a）Particle：Angler bone carried out with high-speed tissue mashing machine to smash processing to pieces, powdery and microparticulate anglers is made
Bone meal；
b）Enzymolysis：Neutral proteinase Dui angler bone meal is selected to digest, Cheng anglers bone meal enzymolysis liquid processed；
c）Ultrafiltration：Ultrafiltration system is added in angler bone meal enzymolysis liquid, ultrafiltration is carried out with the ultrafiltration membrane of 10KD, 5KD, 3KD, is collected
>10KD, 10-5KD, 5-3KD and<The enzymolysis liquid of 3KD molecular weight, is freeze-dried,
d）Angler enzymolysis liquid is crossed into G-25 eluents and obtains 5 peaks, is screened through anticancer experiment in vitro, peak 2 and peak 3 have most
High antitumor activity, collect peak 2 and peak 3, freeze-drying further isolates and purifies and with efficient liquid phase purity detecting；
e）By reversed-phase high performance liquid chromatography (RT- HPLC) purifying and amino acid sequence detection：Peak 2 is detected with HPLC and is found, should
Molecule section only has 1 eluting peak, and carries out amino acid sequence detection, and amino acid sequence is Ala-Pro-Phe-His-Asp-
Ser-Asp-Gln-Asn- Cys；Make into ten peptide product of angler fracture tumour.
2. according to the preparation method of claim 1 Suo Shu angler fracture tumour decapeptides, it is characterised in that：
The step b）Enzymatic hydrolysis condition is：pH:7.0-7.5, temperature：40-45 DEG C, solid-liquid ratio：1:1, the time：5-7h, it is enzyme
Amount:1200-1500 u/g；In the step c, ten peptide product of angler fracture tumour made, amino acid sequence is
Ala-Pro-Phe-His-Asp-Ser-Asp-Gln-Asn- Cys, the step d）In, the G-25 gels separation：It will
Step c）The enzymolysis liquid of acquisition is dissolved with distilled water, and centrifugation takes supernatant, crosses 0.45 μm of miillpore filter, takes sample solution
1.5ml crosses SephadexG-25 columns (cm of 90cm × 115 (ID)), is eluent with distilled water, balance and elution；Often pipe is collected
3ml detects at λ 280nm, collects each peak eluent；
The step e）In, the reversed-phase high performance liquid chromatography (RT- HPLC)：Purified with RT-HPLC, chromatostrip
Part:1) sample pre-treatments:Sample is centrifuged with the water dissolution of 0.06%TFA to 0.6ml（12000-14000RPM centrifuges 10 points
Clock）Take supernatant；2) system: Agilent 1260 HPLC；3) pillar:ZorbaxSB-C18 4.6X250 5um；4) sample introduction body
Product:80-100ul；5) buffer solution, equilibration buffer:0.06%TFA elution buffers:The acetonitrile of 0.05%TFA；6) gradient:
0% -0 % B are eluted 4 minutes, and 0% -8% B is eluted 25 minutes, and 8%-100%B is eluted 1 minute, and 100% -100%B is eluted 5 minutes；7)
Flow velocity:1.0 ml/min；8）Detection:280nm/214nm.
3. a kind of application such as claims 1 or 2 Suo Shu angler fracture tumour decapeptides, it is characterised in that Suo Shu angler bones
Antitumor decapeptide is used for the additional functionality food of patients with prostate cancer, to the inhibiting effect of prostate cancer as functional food
Obviously.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690370A (en) * | 2011-12-20 | 2012-09-26 | 浙江省海洋开发研究院 | Comprehensive utilization technique of marine fish bones |
| CN103805662A (en) * | 2012-11-15 | 2014-05-21 | 浙江海洋学院 | Preparation method and application of sinonovacula constricta enzymolysis polypeptide |
| CN104304647A (en) * | 2014-09-12 | 2015-01-28 | 浙江海洋学院 | Monkfish leftover classification using method |
| CN104774896A (en) * | 2015-04-15 | 2015-07-15 | 浙江海洋学院 | Preparation method for iron-chelated collagen peptide of hairtail fish-bones |
-
2018
- 2018-01-16 CN CN201810040290.0A patent/CN108359702A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690370A (en) * | 2011-12-20 | 2012-09-26 | 浙江省海洋开发研究院 | Comprehensive utilization technique of marine fish bones |
| CN103805662A (en) * | 2012-11-15 | 2014-05-21 | 浙江海洋学院 | Preparation method and application of sinonovacula constricta enzymolysis polypeptide |
| CN104304647A (en) * | 2014-09-12 | 2015-01-28 | 浙江海洋学院 | Monkfish leftover classification using method |
| CN104774896A (en) * | 2015-04-15 | 2015-07-15 | 浙江海洋学院 | Preparation method for iron-chelated collagen peptide of hairtail fish-bones |
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Application publication date: 20180803 |