CN107337716A - The method that biological oxytocin solution is extracted in high-voltage power supply - Google Patents
The method that biological oxytocin solution is extracted in high-voltage power supply Download PDFInfo
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- CN107337716A CN107337716A CN201710547879.5A CN201710547879A CN107337716A CN 107337716 A CN107337716 A CN 107337716A CN 201710547879 A CN201710547879 A CN 201710547879A CN 107337716 A CN107337716 A CN 107337716A
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- oxytocin
- power supply
- voltage power
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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
- C07K7/16—Oxytocins; Vasopressins; Related peptides
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- Genetics & Genomics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention provides a kind of method that biological oxytocin solution is extracted in high-voltage power supply, belong to biomedicine technical field, specifically using the polypeptide moiety in biotechnology extraction posterior pituitary dry powder, separated again by preparative high-voltage power supply, oxytocin ultraviolet spectra is monitored using PDAD in real time in electrophoresis fluid outlet, oxytocin section efflux is collected, finally removes organic colored thing and pyrogen using bentonite, obtains oxytocin solution.This method is simple to operate, high income, the biological oxytocin purity height of extraction, bioactivity height.
Description
Technical field
The present invention relates to biomedicine technical field, the side of biological oxytocin solution is extracted in specifically a kind of high-voltage power supply
Method.
Background technology
Oxytocin（Oxytocin）, also known as oxytocins, be it is a kind of it is being made up of 9 amino acid residues, there are two sulphur
Key, have bioactivity polypeptide product, molecular formula C43H66N12O12S2, molecular weight 1007.2, isoelectric point 7.7 is readily soluble
Yu Shui, be dissolved in acetone, butanol and spirit of vinegar, insoluble in ether and petroleum ether, dry product is stable, pH be 3.5~4.4 it is acid molten
It is stable in liquid, it is unstable in alkaline solution.
It is main at present that oxytocin is prepared using chemical synthesis.But the oxytocin crude product of chemical synthesis contains some and contracting
The similar miscellaneous peptide of palace element structure, such as the not connected upper generation of diastereoisomer, partial amino-acid caused by amino acid racemization
Disappearance peptide, fracture peptide caused by peptide bond fracture and oxidation thoroughly caused by reduction-state open loop peptide etc., cause oxytocin to produce
Product yield is relatively low, purification difficult.And the oxytocin prepared using chemical synthesis can not be formed its natural double S keys β-time
The structure of folding, the bioactivity of product are relatively low.
Compared with chemical synthesis, biological extraction method will not introduce the analogue of oxytocin, such as dimer, diastereomeric
Isomers, fracture peptide, open loop peptide etc., but biological raw material complicated, relatively low turn into always of the content of oxytocin restrict its application
Bottleneck.In addition, the cellulose content that boosted in the oxytocin solution of prior art extraction is generally 5% or so, it is difficult to meets to require.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of method that biological oxytocin solution is extracted in high-voltage power supply, behaviour
It is high to make simple, high income, the oxytocin solution purity obtained height, bioactivity, is adapted to extract biological oxytocin solution on a large scale.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme that：It is molten that biological oxytocin is extracted in high-voltage power supply
The method of liquid, comprises the following steps：
（1）, weigh animal posterior pituitary dry powder and extracting in water, centrifuge and collect centrifugate；
（2）, centrifugate pass through preparative high-voltage power supply separator, the setting rate of outflow is 20 mL/min, separation voltage 15
KV, holder select polyacrylamide gel, and cushioning liquid is to adjust pH value to 3.5~4.0 solution, diode with glacial acetic acid
Permutation detector monitors the characteristic spectrum in the range of the nm of electrophoresis efflux 250~400 in real time, and 120 after electrophoresis starts~
Start to collect electrophoresis efflux when occurring 280 nm absorption peak strengths in 240 min first more than 0.15, after electrophoresis starts
Stop collecting electrophoresis efflux when occurring 280 nm absorption peak strengths in 240~360 min first less than 0.15；
（3）, the electrophoresis efflux glacial acetic acid being collected into adjusted into pH value to 3.5~4.0, be heated to 95~100 DEG C, insulation 15
~30 minutes, cooled and filtered, collect filtrate and adjust pH value to 3.5~4.0 with glacial acetic acid, the bentonite slurry for adding 10% stirs
Absorption 40~60 minutes, filters and collects filtrate, as oxytocin solution.
Step（1）Twice, the total amount and the weight ratio of posterior pituitary dry powder of extraction plus water are extracting in water twice（10
~15）：1, each extraction time is 30~40 min, and extract solution merging twice is stand-by.
Step（3）The addition of bentonite is：Every 100 mL filtrates add 3 mL 10% bentonite slurry.
Then the present invention utilizes preparative high-voltage power supply separator using the oxytocin in water extraction posterior pituitary dry powder
Isolated and purified, the content of vasopressin is minimized, finally recycle bentonite absorption to remove small-molecule substance, organic colored
Thing and pyrogen, so as to obtain the higher oxytocin solution of purity.The extracting method of the present invention is simple to operate, the oxytocin extracted
Not only purity is high for solution, and has higher bioactivity, the cellulose content that can boost in oxytocin solution control 0.5% with
Under, security has a more substantial increase, and suitable for the large-scale production of oxytocin.
Embodiment
With reference to embodiment, the invention will be further described：
Embodiment 1：
The method that biological oxytocin solution is extracted in high-voltage power supply, comprises the following steps：
（1）Take leaf dried powder after pig pituitary（It is commercially available）1000 g, the L of water for injection 5 is added, stirring extraction 30 minutes, is collected by centrifugation
Liquid, residue add water 5 L, stirring extraction 30 minutes, liquid are collected by centrifugation, merges centrifugate twice；
（2）, centrifugate pass through preparative high-voltage power supply separator, the setting rate of outflow is 20 mL/min, and holder is from poly-
Acrylamide gel, cushioning liquid are to balance two electrophoretic apparatus volumes with the solution of glacial acetic acid regulation pH value to 3.5, buffer solution
Afterwards, pour into centrifugate, start shooting separation voltage 15kV, timing and diode permutation detector monitor in real time electrophoresis efflux 250~
Characteristic spectrum in the range of 400 nm, at the 126th minute, 280 nm absorption peak strengths started to be more than 0.15, now started to collect
Electrophoresis efflux；At the 304th minute, 280 nm absorption peak strengths were less than 0.15, stopped collecting electrophoresis efflux；
（3）, the electrophoresis efflux glacial acetic acid being collected into adjusted into pH value to 3.5, water-bath quickly heats up to 100 DEG C, insulation 30
Minute, paper pulp filtering after cooling down immediately, collect filtrate and adjust pH value to 3.5 with glacial acetic acid, 3 are added by every 100 mL filtrates
The dosage of the bentonite of mL 10% slurry adds bentonite, and for stirring and adsorbing after 40 minutes, it is to contract to collect nearly colorless cleared solution after filtering
Palace element solution.Through weighing and calculating, the unit of oxytocin about 1010 is made in leaf dried powder after every gram of pig pituitary.
Embodiment 2：
The method that biological oxytocin solution is extracted in high-voltage power supply, comprises the following steps：
（1）Take leaf dried powder after pig pituitary（It is commercially available）1000 g, the L of water for injection 9 is added, stirring extraction 40 minutes, is collected by centrifugation
Liquid, residue add water 6 L, stirring extraction 40 minutes, liquid are collected by centrifugation, merges centrifugate twice；
（2）, centrifugate pass through preparative high-voltage power supply separator, the setting rate of outflow is 20 mL/min, and holder is from poly-
Acrylamide gel, cushioning liquid are to balance two electrophoretic apparatus volumes with the solution of glacial acetic acid regulation pH value to 3.5, buffer solution
Afterwards, pour into centrifugate, start shooting the kV of separation voltage 15, timing and diode permutation detector monitor in real time electrophoresis efflux 250~
Characteristic spectrum in the range of 400 nm, at the 138th minute, 280 nm absorption peak strengths started to be more than 0.15, now started to collect
Electrophoresis efflux；At the 325th minute, 280 nm absorption peak strengths were less than 0.15, stopped collecting electrophoresis efflux；
（3）, the electrophoresis efflux glacial acetic acid being collected into adjusted into pH value to 3.5, water-bath quickly heats up to 95 DEG C, is incubated 40 points
Clock, paper pulp filtering after cooling down immediately, collect filtrate and adjust pH value to 3.5 with glacial acetic acid, 3 are added by every 100 mL filtrates
The dosage of the bentonite of mL 10% slurry adds bentonite, and for stirring and adsorbing after 50 minutes, it is to contract to collect nearly colorless cleared solution after filtering
Palace element solution.Through weighing and calculating, the unit of oxytocin about 1185 is made in every gram of sheep brain posterior pituitary dry powder.
Embodiment 3：
The method that biological oxytocin solution is extracted in high-voltage power supply, comprises the following steps：
（1）Take sheep brain posterior pituitary dry powder（It is commercially available）1000 g, the L of water for injection 8 is added, stirring extraction 40 minutes, is collected by centrifugation
Liquid, residue add water 5 L, stirring extraction 30 minutes, liquid are collected by centrifugation, merges centrifugate twice；
（2）, centrifugate pass through preparative high-voltage power supply separator, the setting rate of outflow is 20 mL/min, and holder is from poly-
Acrylamide gel, cushioning liquid are to balance two electrophoretic apparatus volumes with the solution of glacial acetic acid regulation pH value to 4.0, buffer solution
Afterwards, pour into centrifugate, start shooting the kV of separation voltage 15, timing and diode permutation detector monitor in real time electrophoresis efflux 250~
Characteristic spectrum in the range of 400 nm, at the 131st minute, 280 nm absorption peak strengths started to be more than 0.15, now started to collect
Electrophoresis efflux；At the 318th minute, 280 nm absorption peak strengths were less than 0.15, stopped collecting electrophoresis efflux；
（3）, the electrophoresis efflux glacial acetic acid being collected into adjusted into pH value to 4.0, water-bath quickly heats up to 95 DEG C, is incubated 40 points
Clock, paper pulp filtering after cooling down immediately, collect filtrate and adjust pH value to 4.0 with glacial acetic acid, 3 are added by every 100 mL filtrates
The dosage of the bentonite of mL 10% slurry adds bentonite, and for stirring and adsorbing after 60 minutes, it is to contract to collect nearly colorless cleared solution after filtering
Palace element solution.Through weighing and calculating, the unit of oxytocin about 1060 is made in every gram of sheep brain posterior pituitary dry powder.
Above example is only intended to help the method and core concept for understanding the present invention.It should be pointed out that for this technology
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
Claims (4)
1. the method that biological oxytocin solution is extracted in high-voltage power supply, it is characterised in that comprise the following steps：
（1）, weigh animal posterior pituitary dry powder and extracting in water, centrifuge and collect centrifugate；
（2）, centrifugate pass through preparative high-voltage power supply separator, the setting rate of outflow is 20 mL/min, separation voltage 15
KV, holder select polyacrylamide gel, and cushioning liquid is to adjust pH value to 3.5~4.0 solution, diode with glacial acetic acid
Permutation detector monitors the characteristic spectrum in the range of the nm of electrophoresis efflux 250~400 in real time, and 120 after electrophoresis starts~
Start to collect electrophoresis efflux when occurring 280 nm absorption peak strengths in 240 min first more than 0.15,240 after electrophoresis starts
Stop collecting electrophoresis efflux when occurring 280 nm absorption peak strengths in~360 min first less than 0.15；
（3）, the electrophoresis efflux glacial acetic acid being collected into adjusted into pH value to 3.5~4.0, be heated to 95~100 DEG C, insulation 15
~30 minutes, cooled and filtered, collect filtrate and adjust pH value to 3.5~4.0 with glacial acetic acid, the bentonite slurry for adding 10% stirs
Absorption 40~60 minutes, filters and collects filtrate, as oxytocin solution.
2. the method that biological oxytocin solution is extracted in high-voltage power supply according to claim 1, it is characterised in that：Step（1）
Twice, each extraction time is 30~40 min to extracting in water, and extract solution merging twice is stand-by.
3. the method that biological oxytocin solution is extracted in high-voltage power supply according to claim 2, it is characterised in that：Step（1）
The total amount and the weight ratio of posterior pituitary dry powder of extraction plus water are twice（10~15）：1.
4. the method that biological oxytocin solution is extracted in high-voltage power supply according to claim 1, it is characterised in that：Step（3）
The addition of bentonite is：Every 100 mL filtrates add 3 mL 10% bentonite slurry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710547879.5A CN107337716A (en) | 2017-07-06 | 2017-07-06 | The method that biological oxytocin solution is extracted in high-voltage power supply |
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| CN201710547879.5A CN107337716A (en) | 2017-07-06 | 2017-07-06 | The method that biological oxytocin solution is extracted in high-voltage power supply |
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| CN107337716A true CN107337716A (en) | 2017-11-10 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087152A (en) * | 2013-01-17 | 2013-05-08 | 南京新百药业有限公司 | Extraction process of oxytocin solution |
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- 2017-07-06 CN CN201710547879.5A patent/CN107337716A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087152A (en) * | 2013-01-17 | 2013-05-08 | 南京新百药业有限公司 | Extraction process of oxytocin solution |
Non-Patent Citations (1)
| Title |
|---|
| 段永红等: "《遗传标记在植物研究中的应用》", 31 May 2010, 中国农业科学技术出版社 * |
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Application publication date: 20171110 |