CN107434818A - A kind of separation method of antler polypeptide - Google Patents
A kind of separation method of antler polypeptide Download PDFInfo
- Publication number
- CN107434818A CN107434818A CN201510914036.5A CN201510914036A CN107434818A CN 107434818 A CN107434818 A CN 107434818A CN 201510914036 A CN201510914036 A CN 201510914036A CN 107434818 A CN107434818 A CN 107434818A
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- China
- Prior art keywords
- antler
- peak activity
- freezed
- purifies
- hac
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
Abstract
The invention discloses a kind of method that antler polypeptide isolates and purifies, methods described includes：Fresh pilose antler after flushing is crushed, and adds homogenate；Fresh pilose antler powder after addition homogenate is subjected to centrifugal treating, takes supernatant, rotary evaporation in vacuo obtains pilose antler concentrate, and pilose antler concentrate is freezed and obtains deer antler extract；Deer antler extract is dissolved, and the deer antler extract after dissolving is chromatographed, obtains Peak Activity eluent, is freezed；The lyophilized Peak Activity eluent is chromatographed, Peak Activity is collected and freezes；Anti-phase high phase liquid chromatography is carried out to the Peak Activity, and collects Peak Activity, is freezed, the antler polypeptide purified.
Description
Technical field
The present invention relates to the method that antler polypeptide isolates and purifies.
Background technology
In the last few years, biological educational circles found that many tissues can itself secretion growth factor successively.The isolated EGFs from mouse submandibular gland of Cohen in 1962；1977, Klagsbrun etc. was purified with the peptides growth factor for promoting chondrocyte proliferation activity from crops osteodiastasis.We isolate and purify obtained polypeptide from red deer pilose antler, proved by detection, promote epidermal cell and the activity of cartilage cell and hepatocyte growth with stronger, but, because we can not determine that it is natural free existing growth factor or artificial extraction product at present, therefore temporarily it is called antler polypeptide.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of method that antler polypeptide isolates and purifies.
The purpose of the present invention is realized by following technical scheme：
A kind of method that antler polypeptide isolates and purifies, this method include：
A crushes the fresh pilose antler after flushing, and adds homogenate；
Fresh pilose antler powder after adding homogenate is carried out centrifugal treating by B, takes supernatant, rotary evaporation in vacuo obtains pilose antler concentrate, and pilose antler concentrate is freezed and obtains deer antler extract；
C is dissolved to deer antler extract, and the deer antler extract after dissolving is chromatographed, and obtains Peak Activity eluent, is freezed；
D is chromatographed the lyophilized Peak Activity eluent, is collected Peak Activity and is freezed；
E carries out anti-phase high phase liquid chromatography to the Peak Activity in the step D, and collects Peak Activity, freezes, the antler polypeptide purified.
Compared with prior art, one or more embodiments of the invention can have the following advantages that：
Antler polypeptide is in pale yellow powder shape, soluble in water, and its protein content is 46.7%.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with drawings and Examples, the present invention is described in further detail.
The method isolated and purified as shown in Figure 1 for antler polypeptide, this method include：
Step 10 crushes the fresh pilose antler after flushing, and adds homogenate；
Above-mentioned homogenate is PH3.5 glacial acetic acid aqueous solution.
Fresh pilose antler powder after adding homogenate is carried out centrifugal treating by step 20, takes supernatant, rotary evaporation in vacuo obtains pilose antler concentrate, and pilose antler concentrate is freezed and obtains deer antler extract；
The step specifically includes：Homogenate is subjected to centrifugation 25min with 8000rpm, takes supernatant；
96% ethanol is added in supernatant makes its concentration reach 70%, and centrifugal treating is carried out after deposit 3 hours under the conditions of 5 DEG C, extracts supernatant, to the supernatant rotary evaporation in vacuo of extraction to concentrate, freezes and obtains deer antler extract.
Step 30 is dissolved to deer antler extract, and the deer antler extract after dissolving is chromatographed, and obtains Peak Activity eluent, is freezed；
With PH4.5 HAc-NaAc
Buffer solution fully balances, and is added sample in post with 3ml/min flow velocitys, and use PH4.5 HAc-NaAc respectively with 12ml/min flow velocity
Buffer solution, PH5.0 HAc-NaAc
Buffer solution elutes, and collects Peak Activity eluent；
Eluent is dialysed with bag filter, and freezed.
Step 40 is chromatographed the lyophilized Peak Activity eluent, is collected Peak Activity and is freezed；
With PH5.0 HAc-NaAc buffer solutions；
Load PH5.0 HAc-NaAc
Buffer solution is fully balanced, and sample is added in post, is eluted with PH5.0 HAc-NaAc buffer solutions, and flow velocity is that 2ml/min collects Peak Activity, and is freezed.
Step 50 carries out anti-phase high phase liquid chromatography to the Peak Activity in the step 40, and collects Peak Activity, freezes, the antler polypeptide purified.
Dried frozen aquatic products is dissolved with distilled water, and is filtered, is fully balanced with isopropyl alcohol and water, the ratio of its isopropyl alcohol and water is 0.42：0.7, sample size is 15mg/ml 0.1ml, elution, flow velocity 2ml/min, Detection wavelength 280nm；Peak Activity is collected, is freezed.
Above-described embodiment is crushed Pilose Antler Tissues using crush method, and makes its clasmatosis by colloid mill, so ensures that intracellular matter and intercellular substance are extracted.
Although disclosed herein embodiment as above, the description is merely the mode of execution for facilitating the understanding of the present invention, is not limited to the present invention.Any those skilled in the art to which this invention pertains; do not depart from disclosed herein spirit and scope on the premise of; any modification and change can be made in the implementing form and in details; but the scope of patent protection of the present invention, still should be subject to the scope of the claims as defined in the appended claims.
Claims (6)
1. a kind of method that antler polypeptide isolates and purifies, it is characterised in that methods described includes：
A crushes the fresh pilose antler after flushing, and adds homogenate；
Fresh pilose antler powder after adding homogenate is carried out centrifugal treating by B, takes supernatant, rotary evaporation in vacuo obtains pilose antler concentrate, and pilose antler concentrate is freezed and obtains deer antler extract；
C is dissolved to deer antler extract, and the deer antler extract after dissolving is chromatographed, and obtains Peak Activity eluent, is freezed；
D is chromatographed the lyophilized Peak Activity eluent, is collected Peak Activity and is freezed；
E carries out anti-phase high phase liquid chromatography to the Peak Activity in the step D, and collects Peak Activity, freezes, the antler polypeptide purified.
2. the method that antler polypeptide according to claim 1 isolates and purifies, it is characterised in that homogenate is PH3.5 glacial acetic acid aqueous solution in the step A.
3. the method that antler polypeptide according to claim 1 isolates and purifies, it is characterised in that the step B is specifically included：Homogenate is subjected to centrifugation 25min with 8000rpm, takes supernatant；
96% ethanol is added in supernatant makes its concentration reach 70%, and centrifugal treating is carried out after deposit 3 hours under the conditions of 5 DEG C, extracts supernatant, to the supernatant rotary evaporation in vacuo of extraction to concentrate, freezes and obtains deer antler extract.
4. the method that antler polypeptide according to claim 1 isolates and purifies, it is characterised in that the step C is specifically included：
With PH4.5 HAc-NaAc
Buffer solution fully balances, and is added [a1] in post with 3ml/min flow velocitys, and is eluted respectively with PH4.5 HAc-NaAc buffer solutions, PH5.0 HAc-NaAc buffer solutions with 12ml/min flow velocity, collects Peak Activity eluent；
Eluent is dialysed with bag filter, and freezed.
5. the method that antler polypeptide according to claim 1 isolates and purifies, it is characterised in that the step D is specifically included：With PH5.0 HAc-NaAc buffer solutions；
Filling PH5.0 HAc-NaAc buffer solutions are fully balanced, and [a2] is added in post, are eluted with PH5.0 HAc-NaAc buffer solutions, and flow velocity is that 2ml/min collects Peak Activity, and is freezed.
6. the method that antler polypeptide according to claim 1 isolates and purifies, it is characterised in that the step E is specifically included：Dried frozen aquatic products is dissolved with distilled water, and is filtered, is fully balanced with isopropyl alcohol and water, the ratio of its isopropyl alcohol and water is 0.42：0.7, sample size is 15mg/ml 0.1ml, elution, flow velocity 2ml/min, Detection wavelength 280nm；Peak Activity is collected, is freezed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510914036.5A CN107434818A (en) | 2016-05-25 | 2016-05-25 | A kind of separation method of antler polypeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510914036.5A CN107434818A (en) | 2016-05-25 | 2016-05-25 | A kind of separation method of antler polypeptide |
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| CN107434818A true CN107434818A (en) | 2017-12-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510914036.5A Pending CN107434818A (en) | 2016-05-25 | 2016-05-25 | A kind of separation method of antler polypeptide |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331976A (en) * | 2000-07-06 | 2002-01-23 | 长春中医学院附属医院 | Traumtin prepn for injecta and its prepn process |
| CN101020715A (en) * | 2007-03-15 | 2007-08-22 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
-
2016
- 2016-05-25 CN CN201510914036.5A patent/CN107434818A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331976A (en) * | 2000-07-06 | 2002-01-23 | 长春中医学院附属医院 | Traumtin prepn for injecta and its prepn process |
| CN101020715A (en) * | 2007-03-15 | 2007-08-22 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
Non-Patent Citations (2)
| Title |
|---|
| 梅兵等: "鹿茸多肽提取分离及药理活性的研究进展", 《中华中医药杂志》 * |
| 陶荣珊等: "鹿茸多肽提取分离纯化及药理作用研究进展", 《经济动物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
| CN113278047B (en) * | 2021-06-28 | 2023-05-12 | 上海中盈经济发展(集团)有限公司 | Processing method for extracting compound active polypeptide from fresh pilose antler |
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Application publication date: 20171205 |
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