CN105998076A - Submandibular gland active molecule separation and extraction process and active molecule product thereof - Google Patents
Submandibular gland active molecule separation and extraction process and active molecule product thereof Download PDFInfo
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- CN105998076A CN105998076A CN201610573535.7A CN201610573535A CN105998076A CN 105998076 A CN105998076 A CN 105998076A CN 201610573535 A CN201610573535 A CN 201610573535A CN 105998076 A CN105998076 A CN 105998076A
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- bioactive molecule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/55—Glands not provided for in groups A61K35/22 - A61K35/545, e.g. thyroids, parathyroids or pineal glands
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention discloses a submandibular gland active molecule separation and extraction process and an application method thereof. The problem that existing operation method for obtaining single active molecules are not suitable for industrialized production and are high in input cost is solved. The submandibular gland active molecule separation and extraction process comprises the steps that step one, a submandibular gland is obtained, a buffer solution is added, and then a submandibular gland extracting solution is obtained through crushing homogenate; step two, a ceramic filter membrane is adopted to perform more than one time of filtration, a preliminary filtering solution is collected, and then active molecule filtrate is obtained after an organic membrane is adopted for filtration. The invention further discloses an active molecule product prepared by adopting the active molecule filtrate. The submandibular gland active molecule separation and extraction process has the advantages of being suitable for industrialized production, low in cost, remarkable in effect and the like.
Description
Technical field
The present invention relates to the extraction process of a kind of bioactive molecule, be specifically related to a kind of submaxillary gland bioactive molecule and separate
Extraction process, and the bioactive molecule goods that the bioactive molecule solution providing the acquisition of this technique is made.
Background technology
In prior art, the extraction of intracellular reactive molecule is all individually to extract one type, is then inciting somebody to action
This class bioactive molecule extracted adds in biological product, and then the physics and chemistry reaching such active factors is made
With.
The extraction of existing intracellular reactive molecule is required to operate through organic membrane filter and chromatographic column chromatography etc.
Method, thus when using existing method to obtain single-activity molecule, its operational approach is not suitable for industry metaplasia
Producing, input cost is higher.
Summary of the invention
The technical problem to be solved is: when using existing method to obtain single-activity molecule, its behaviour
Not being suitable for industrialized production as method, input cost is higher.It is an object of the invention to provide and be applicable to work
Industry metaplasia is produced and a kind of submaxillary gland bioactive molecule separation-extraction technology with low cost, and provides and utilize this work
A kind of bioactive molecule goods that the bioactive molecule that skill obtains is made.
The present invention is achieved through the following technical solutions:
A kind of submaxillary gland bioactive molecule separation-extraction technology, including:
Step one, acquisition submaxillary gland, twist homogenate into pieces and obtain submaxillary gland extracting solution after adding buffer；
After step 2, employing ceramic filtration membrane carry out once above filtration, collect preliminary filtrate, adopt the most again
With obtaining bioactive molecule filtrate after organic membrane filter.
The invention provides the acquisition thinking of a kind of new bioactive molecule, be the most all obtain in cell single
A kind of bioactive molecule, the present invention changes original thinking, use the inventive method can obtain include multiple
The bioactive molecule filtrate of active factors.That is, after submaxillary gland extracting solution being carried out initial gross separation by ceramic filtration membrane,
Carry out fine separation by organic membrane again, and then obtain the bioactive molecule filtrate including the various active factor,
Finally by Freeze Drying Technique, the bioactive molecule filtrate including the various active factor can be dried,
Bioactive molecule finished product can be obtained after drying.
By the way of the present invention uses ceramic filtration membrane and organic membrane to be combined filtration separation, submaxillary gland is homogenized
Most of impurity in liquid can be removed, and the inventive method is applicable to industrialized production, improves extraction
Efficiency.And, the present invention is easy to operate, it is to avoid the use of organic solvent, and avoids existing skill
In art, single-activity molecule separates needs to use organic membrane to separate and the method for chromatographic column chromatography, and then makes extraction
Preparation cost is substantially reduced, and ceramic filtration membrane can clean use repeatedly, significantly reduces production cost.And
And, to learn through detection, the effect of the bioactive molecule filtrate obtained by the inventive method is divided with single-activity
Son is compared, and its effect is more outstanding.
Further, the specification of described ceramic filtration membrane is 30KDa～100KDa molecular cut off, and after once make
The molecular cut off of ceramic filtration membrane less than the molecular cut off of front nonrecoverable ceramic filtration membrane；Described have
The specification of machine film is 1～10KDa molecular cut off.
By being preferably provided with of ceramic filtration membrane in the present invention and organic membrane, separation efficiency can be effectively improved, simultaneously
Ensure the best results of the bioactive molecule filtrate extracted.
In order to ensure the activity of bioactive molecule, in described step one, the work of submaxillary gland extracting solution acquisition process
Temperature is 4 DEG C；In described step 2, the operating temperature of filter process controls at 15 DEG C～20 DEG C, pressure control
System is at 0.05Mpa～0.3Mpa.
Further, described submaxillary gland adds buffer after need to rejecting blood vessel, nerve, connective tissue,
This buffer is the acetate buffer solution of 0.05M.
A kind of bioactive molecule goods, are mixed by bioactive molecule filtrate, high molecular weight protein and lyophilizing figuration protective agent
Preparing after closing lyophilizing, bioactive molecule filtrate is made up of Claims 1 to 4 any one technique；This bioactive molecule system
In product, the mass percent of bioactive molecule is 10～15%, the protectant matter of lyophilizing figuration in these bioactive molecule goods
Amount percent is 1～10%.
Can directly be proved by the correction data in table 1: by bioactive molecule and the macromole egg of said ratio
After white and lyophilizing figuration protective agent mixes, the effect of bioactive molecule goods, and effect ten can be greatly enhanced
Clearly demarcated aobvious.
Further, the acquisition methods of described high molecular weight protein is: obtain submaxillary gland, twists into pieces after adding buffer
Homogenate obtains submaxillary gland extracting solution, filters with the defecator of 100KDa～300KDa molecular cut off.
Further, described lyophilizing figuration protective agent includes the one in trehalose, glycine and mannitol
Or it is several.
It is in order to be able to make to merge between high molecular weight protein and bioactive molecule to be more prone to, more uniform in order to be able to mixing,
And few lyophilizing required time can effectively drop；After described high molecular weight protein mixes with bioactive molecule filtrate, pass through egg
White dilution buffer is diluted to the target protein solution that concentration is 1～10mg/ml, slow with albumen dilution the most again
Rush liquid and lyophilizing figuration protective agent is diluted to figuration solution, finally target protein solution is mixed with figuration solution
Postlyophilization.
Further, described albumen dilution buffer is 10～100mM phosphate buffer and percent mass
The solution made after mixing than the arginine being 1%～5%.
The present invention compared with prior art, has such advantages as and beneficial effect:
1, the invention provides a kind of bioactive molecule newly extracts thinking, under this thinking, and the extraction of bioactive molecule
Technique is more applicable for industrialized production, and production efficiency is high, low cost, and extracts the bioactive molecule effect obtained
Fruit is notable；
2, the coordinating of high molecular weight protein and bioactive molecule in the present invention, the effect mutually promoted can be reached, and then
The bioactive molecule goods making the present invention are better.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment, right
The present invention is described in further detail, and the exemplary embodiment of the present invention and explanation thereof are only used for explaining this
Invention, not as a limitation of the invention.
Embodiment 1
The preparation of submaxillary gland extracting solution: take submaxillary gland from adult sheep or fetal lamb, separates the component of organization of attachment,
Shred with shears, carefully reject the blood vessel in body of gland, nerve, connective tissue etc., add the acetic acid of 0.05M
Buffer, twists into pieces, is homogenized three times by homogenizer the most again and obtains submaxillary gland extracting solution, and above step is all at 4 DEG C
Operate under environment.
Submaxillary gland extracting solution is filtered: 10L submaxillary gland extracting solution is filtered through ceramic filtration membrane, pottery
Filter membrane specification is 100KDa molecular cut off, carries out with the distilled water of ceramic filtration membrane filtrate 30% volume
Clean, obtain filtrate and cleanout fluid amounts to 10.3L；Again filtrate and cleanout fluid are passed through 30KDa ceramic filtration membrane,
It is carried out with the distilled water of filtrate 30% volume, obtains filtrate and cleanout fluid adds up to 8.7L.Again will filter
Liquid and cleanout fluid are by organic membrane filter, and organic membrane specification is 1KDa molecular cut off, uses organic membrane filter liquid
The distilled water of 15% volume cleans, and obtains filtrate and cleanout fluid adds up to 7.5L.Again filtrate is passed through 10KDa
Organic membrane carry out retaining concentration, it is thus achieved that molecular cut off is the 4.2L bioactive molecule filtrate of 3～7kD, with
Upper step all 15 DEG C～20 DEG C, operate in the environment of 0.05Mpa～0.3Mpa.
Embodiment 2
The purpose of the present embodiment is to provide one and utilizes bioactive molecule filtrate in embodiment 1 to make bioactive molecule system
The concrete preparation method of product, concrete preparation process is as follows:
The acquisition of high molecular weight protein: use the submaxillary gland extracting solution in embodiment 1, retain down with defecator
The filtrate of 100KDa～300KDa molecular weight, this filtrate is high molecular weight protein.
The preparation of bioactive molecule goods: the bioactive molecule filtrate in embodiment 1 is mixed all with high molecular weight protein
Even, then it is diluted to, by albumen dilution buffer, the target protein solution that concentration is 1～10mg/ml；Use albumen
Lyophilizing figuration protective agent is diluted to figuration solution by dilution buffer；Target protein solution is mixed with figuration solution
After conjunction, bioactive molecule goods are i.e. made in lyophilizing.
Described albumen dilution buffer is 10～100mM phosphate buffers and mass percent is 1%～5%
The solution made after arginine mixing.In the present embodiment, this albumen dilution buffer is by 50mM phosphate buffer
And account for the arginine composition of the mass percent 3% of albumen dilution buffer.
In above-mentioned bioactive molecule goods, the mass percent of bioactive molecule is 10～15%, in these bioactive molecule goods
The protectant mass percent of lyophilizing figuration is 1～10%.Described lyophilizing figuration protective agent includes trehalose, sweet
One or more in propylhomoserin and mannitol.In the present embodiment, this bioactive molecule accounts for bioactive molecule goods gross mass
12%, this lyophilizing figuration protective agent accounts for the 5% of bioactive molecule goods gross mass, and this lyophilizing figuration protective agent
It is chosen as trehalose.
Embodiment 3
The present embodiment is to make lyophilized powder after the bioactive molecule filtrate lyophilizing in embodiment 1, specifically prepares work
Skill is as follows: after this bioactive molecule filtrate is degerming, uses freeze drying process, by concentrated solution temperature-50 DEG C
Under the conditions of lyophilization 10 hours, obtain lyophilized powder.
Embodiment 4
The present embodiment is that sample is carried out epidermal cell proliferation activity influence, and to vascular endothelial cell
(HUVEC) propagation, the adherent and detection of migration impact.Sample in the present embodiment includes embodiment 1 and 2
Finished product, epidermal growth factor and the collagen protein made.
One, the detection of epidermal cell proliferation activity influence
1. epidermis cell is drawn materials and cultivates:
Peritomizing postoperative specimen, PBS washs three times, and eye scissors is rejected more than subcutaneous tissue；25%trypsin
Cold digestion overnight, separates epidermis and corium, and removes corium, prepare cell suspension；1000rpm×10min
Centrifugal, remove supernatant, culture medium suspendible cell again precipitates, and counts with blood-counter system, and expects orchid with platform
Dyeing, understands cell survival rate.Above-mentioned cell suspension is inoculated in culture dish by certain cell density, puts
37 DEG C, 5%CO2In incubator；Within 2 days～3 days, change liquid once；When cell covers with bottle ware 70～80%, according to need
Pass on or freeze-stored cell.
The propagation of 2.MTT method mensuration cell:
Take the logarithm trophophase cell 1.5 × 105It is inoculated in 96 well culture plates, every hole 100 μ l, if 6 multiple
Hole.Add various test sample and zeroing hole adds 100 μ l culture fluid, continue to cultivate 48h, terminate front 4h, add
10 μ l MTT, conventional termination is tested, and surveys each hole light absorption value, wavelength 490nm on enzyme-linked immunosorbent assay instrument.
By the following equation relative rate of increase of calculating: RGR=dosing holes absorbance/control wells absorbance × 100%.
Two, vascular endothelial cell (HUVEC) propagation, the adherent and detection of migration impact
The cell of 1.HUVEC is cultivated
HUVEC cell is the positive through identified by immunofluorescence, eNOS and vWF.Experiment cell used is 6-8
In generation, all culture bottle/plate/wares are coated with 0.2% gelatin in advance.Culture medium is containing 20% hyclone, 60mg/L
Endothelial cell growth supplement and the M199 of 0.005U/L heparin.HUVEC is put 37 DEG C, 5%CO2 incubator
Interior quiescent culture.
2. the sample impact on HUVEC growing multiplication
Cell is inoculated in 24 orifice plates, treat adherent well, grow to 60%-70% saturated time, train with serum-free
Nutrient solution processes 24h, is subsequently adding different sample treatment 2d (matched group adds equivalent PBS), trypsinization, in vain
Cell counting count board counting cell, observes the impact that HUVEC is bred by sample.Often group sets 3 holes, is repeated 3 times.
Use [simultaneously3H] thymidine mixes the impact that synthesizes HUVEC cell DNA of laboratory observation sample.
By 4 × 104HUVEC cell is inoculated in 24 orifice plates, until grow to 60%-70% saturated time, add the most same
Product process cell 24h, and matched group adds equivalent PBS.4h addition before experiment terminates [3H] thymidine
(3.7×103Bq/L), [3H] thymidine will be incorporated in newly synthesized DNA.With cold at the end of experiment
PBS rinsing cell 3 times, addition 0.5mL10% trichloroacetic acid, 4 DEG C of process 2h, 95% ethanol rinse 1 time,
Add 0.2mol/L NaOH lysis at room temperature cell, collect cell, add to 7mL scintillation solution, mixing, liquid
Measure on body scintillation counter [3H] activity (counts/min), calculate suppression ratio.Often group sets 3 holes, weight
Multiple 3 times.
3. sample is on impact adherent for HUVEC
After sample treatment HUVEC 2d (matched group adds equivalent PBS), with trypsin digestion cell, count cell.
Cell (1 × 105) by equivalent is inoculated in the culture dish of the 35mm that fibronectin (Sigma) was coated, and cultivates
After 30min, the most adherent cell PBS is washed away, fixes 40min with the paraformaldehyde of 3.7%, then use
0.1% violet staining 40min, 10% acetic acid decolouring, 570nm colorimetric, observe sample adherent to HUVEC
Impact.This experiment is independently carried out 3 times.
4. the impact that HUVEC is migrated by sample
According to document, by scratching the impact on endothelial migration of the laboratory observation sample.By equivalent HUVEC kind extremely
The culture dish of 35mmol/L, cultivation to 90% is saturated.Add variable concentrations sample treatment HUVEC2d.Use 1mL
Sample-adding suction nozzle in the middle of culture dish, scratch 2 road wounds respectively, PBS rinses 2 times, add fresh medium and
Sample, the cut distance before computation migration of taking pictures.Continuing to cultivate, after scratching, 20h takes pictures respectively, calculates
Machine software draws 2 curves along cell edges, calculates the average distance between 2 curves, then calculates relatively
Mobility: relative mobility=(average distance of original width-20h)/original width × 100%, observes sample pair
The impact that HUVEC migrates.This experiment is independently carried out 3 times.
Sample in the present embodiment is to epidermal cell proliferation activity influence, and to vascular endothelial cell (HUVEC)
Propagation, adherent and migration impact testing result are as shown in table 1.
Table 1
Matched group is without case of comparative examples during sample, the data by table 1: the inventive method
The effect of the bioactive molecule filtrate obtained is compared with single-activity molecule, and its effect is more outstanding.And pass through
After the bioactive molecule of the invention described above and high molecular weight protein and lyophilizing figuration protective agent mix, can greatly carry
The effect of high activity molecular product, and effect is fairly obvious.
Above-described detailed description of the invention, is carried out the purpose of the present invention, technical scheme and beneficial effect
Further describe, be it should be understood that the detailed description of the invention that the foregoing is only the present invention,
The protection domain being not intended to limit the present invention, all within the spirit and principles in the present invention, that is done is any
Amendment, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (9)
1. a submaxillary gland bioactive molecule separation-extraction technology, it is characterised in that including:
Step one, acquisition submaxillary gland, twist homogenate into pieces and obtain submaxillary gland extracting solution after adding buffer；
After step 2, employing ceramic filtration membrane carry out once above filtration, collect preliminary filtrate, adopt the most again
With obtaining bioactive molecule filtrate after organic membrane filter.
A kind of submaxillary gland bioactive molecule separation-extraction technology the most according to claim 1, it is characterised in that
The specification of described ceramic filtration membrane is the molecular cut off of 30KDa～100KDa, and rear nonrecoverable pottery
The molecular cut off of porcelain filter membrane is less than the molecular cut off of front nonrecoverable ceramic filtration membrane；Described organic membrane
Specification is the molecular cut off of 1～10KDa.
A kind of submaxillary gland bioactive molecule separation-extraction technology the most according to claim 1, it is characterised in that
In described step one, the operating temperature of submaxillary gland extracting solution acquisition process is 4 DEG C；In described step 2,
The operating temperature of filter process controls at 15 DEG C～20 DEG C, and Stress control is at 0.05Mpa～0.3Mpa.
A kind of submaxillary gland bioactive molecule separation-extraction technology the most according to claim 1, it is characterised in that
Described submaxillary gland adds buffer after need to rejecting blood vessel, nerve, connective tissue, and this buffer is 0.05M
Acetate buffer solution.
5. bioactive molecule goods, it is characterised in that by bioactive molecule filtrate, high molecular weight protein and freeze
Preparing after dry figuration protective agent is freeze-dried mixed, bioactive molecule filtrate is made up of Claims 1 to 4 any one technique；
In these bioactive molecule goods, the mass percent of bioactive molecule is 10～15%, and in these bioactive molecule goods, lyophilizing is composed
The protectant mass percent of shape is 1～10%.
A kind of bioactive molecule goods the most according to claim 5, it is characterised in that
The acquisition methods of described high molecular weight protein is: obtain submaxillary gland, twists homogenate into pieces and obtain after adding buffer
Submaxillary gland extracting solution, the defecator of 100KDa～300KDa molecular cut off obtains after filtering.
A kind of bioactive molecule goods the most according to claim 5, it is characterised in that
Described lyophilizing figuration protective agent includes one or more in trehalose, glycine and mannitol.
A kind of bioactive molecule goods the most according to claim 5, it is characterised in that
After described high molecular weight protein mixes with bioactive molecule filtrate, it is diluted to concentration by albumen dilution buffer
It is the target protein solution of 1～10mg/ml, the most again by albumen dilution buffer by dilute for lyophilizing figuration protective agent
It is interpreted into figuration solution, finally by target protein solution and figuration solution mixing postlyophilization.
A kind of bioactive molecule goods the most according to claim 8, it is characterised in that
Described albumen dilution buffer is 10～100mM phosphate buffers and mass percent is 1%～5%
The solution made after arginine mixing.
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| CN201610573535.7A CN105998076A (en) | 2016-07-20 | 2016-07-20 | Submandibular gland active molecule separation and extraction process and active molecule product thereof |
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| CN201610573535.7A CN105998076A (en) | 2016-07-20 | 2016-07-20 | Submandibular gland active molecule separation and extraction process and active molecule product thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682765A (en) * | 2005-03-15 | 2005-10-19 | 江中药业股份有限公司 | Method for refining sheep placenta active component using membrane separating technology |
| CN101613394A (en) * | 2008-06-27 | 2009-12-30 | 熊玲媛 | The preparation method of the preparation method of mouse nerve growth factor and injection mouse nerve growth factor |
-
2016
- 2016-07-20 CN CN201610573535.7A patent/CN105998076A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682765A (en) * | 2005-03-15 | 2005-10-19 | 江中药业股份有限公司 | Method for refining sheep placenta active component using membrane separating technology |
| CN101613394A (en) * | 2008-06-27 | 2009-12-30 | 熊玲媛 | The preparation method of the preparation method of mouse nerve growth factor and injection mouse nerve growth factor |
Non-Patent Citations (1)
| Title |
|---|
| 陈子琏等: "《人体结构学》", 31 January 2001, 科学出版社 * |
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