CN105617350A - Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof - Google Patents
Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof Download PDFInfo
- Publication number
- CN105617350A CN105617350A CN201610076074.2A CN201610076074A CN105617350A CN 105617350 A CN105617350 A CN 105617350A CN 201610076074 A CN201610076074 A CN 201610076074A CN 105617350 A CN105617350 A CN 105617350A
- Authority
- CN
- China
- Prior art keywords
- sheep
- mass fraction
- sclera
- scleral collagen
- collagen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
- A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
-
- 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
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- 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
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Ophthalmology & Optometry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Neurosurgery (AREA)
- Biotechnology (AREA)
- Neurology (AREA)
- Virology (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Preparation (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention particularly relates to an extraction method of sheep sclera collagen and a preparation method of ophthalmic gel. An enzyme hydrolysis-salt extraction method is adopted to extract collagen from sheep eye sclera. The invention further provides the ophthalmic gel with the sheep sclera collagen. The ophthalmic gel comprises, by mass, 80-90% of the sheep sclera collagen, 0.01-2% of preservative,0.5-2% of boric acid and borax, 0.5-2% of polyvinyl alcohol, 0.3-2% of Tween, 0.5-2% of sodium chloride and the balance of water for injection. The sheep sclera collagen is similar to human eye sclera collagen in structure and has the advantages of high purity, high tissue compatibility, low molecular weight and low repellency.
Description
Technical field
The present invention relates to pharmaceutical technology field, particularly relate to the extracting method of a kind of sheep scleral collagen, gel for eye use and preparation method thereof.
Background technology
Collagen protein belongs to macro-molecular protein, and its ultrastructure unit is collagen fiber, owing to the polymerization methods of each collagen type is different, determines they Various Functions. People's eye sclera is made up of extracellular matrix and a small amount of cell, and collagen fiber are the important component parts of extracellular matrix, account for the 75% of sclera dry weight. The diameter of shallow-layer sclera collagen fiber is 166nm, and the diameter of Deep Layer of Sclera collagen fiber is 100nm. Different parts at eyeball, the mode of collagenous fiber bundle arrangement is different: look around nipple at the anterior nearly limbus of corneae place of eyeball, eyeball rear portion, the position of sclera is penetrated including arteriae ciliares posteriores breves and nerve, collagenous fiber bundle circular array, small part is netted, is adapted as the fulcrum of power, extraocular muscles attachment; From the anterior nearly limbus of corneae of eyeball to the nearly cribriform plate of sclera at eyeball rear portion, the general trend of collagenous fiber bundle arrangement is fore-and-aft direction (meridian direction), and wherein Posterior pole sclera reticular fiber is more, has certain expansivity. Additionally, collagen fiber make sclera have very obdurability, play an important role for maintaining eyeball shape configuration aspects in antagonism intraocular pressure therefore collagen. When Posterior pole collagen protein deficiency, posterior pole of eyeball portion arrangement of collagen fibers gets muddled, and sclera there will be toughness to be reduced, and expansivity increases, and eyeball is had a negative impact. It is to say, collagen protein is to maintain eyeball normal morphology, axis oculi normal length, the sclera indispensable important component of normal thickness, is prevent posterior pole of eyeball portion sclera from extending further, and thickness is thinning further, the material that posterior scleral staphyloma occurs. Therefore, exogenous replenishing collagen can pre-myopic-preventing further development, the treatment of pathologic myopia is very helpful.
Collagen protein is that people's in-vivo content is the abundantest, is distributed one group of scleroprotein the most widely, it has many biological characteristicses such as low immunogenicity, biodegradability and promoting growth of cell, various oculopathy can be treated clinically as pharmaceutical carrier, promote the reparation of eye corneal epithelium damage and the healing of corneal wound, be a kind of good eye medicinal preparation. But currently used collagen protein great majority come from the extract of animal tissue, skin etc., such as the skin of animal, tendon, cartilage, scale etc., there is infection disease, cause the defects such as rejection, molecular weight are big.
Summary of the invention
For defect problems such as overcoming ophthalmically acceptable collagen protein in prior art to have infection disease, cause rejection, molecular weight big, the present invention utilizes enzyme hydrolysis-salt extraction method, provide a kind of collagen protein that extracts from sheep sclera and prepare sheep scleral collagen gel for eye use, including sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 80%��90%, the mass fraction of preservative is 0.01%��2%, the mass fraction of boric acid and Borax is 0.5%��2%, the mass fraction of polyvinyl alcohol is 0.5%��2%, the mass fraction of sodium chloride is 0.5��2%, and the mark of tween quality is 0.3%��2%, and the mass fraction of water for injection is surplus.
Wherein, the extracting method of described sheep scleral collagen comprises the following steps:
1) in vitro sheep eyeball is removed other parts except sclera, isolate sheep sclera;
2) by step 1) in the sheep sclera that obtains be cut into sheep sclera sheet; With normal saline soaking and washing sheep sclera sheet, after removing serum and fat, rinse with distilled water, dry in an aseptic environment, obtain sheep sclera dry tablet;
3) taking described sheep sclera dry tablet, add pepsin, described pepsic quality is the 10% of sheep sclera dry tablet quality, puts into after refiner carries out homogenate, is placed on magnetic stirrer stirring and extracts 24h��26h;
4) being 0��4 DEG C with High speed refrigerated centrifuge in temperature, when rotating speed is 6000 turns/min, centrifugal 10��30min, takes first time supernatant solution, and is precipitated;
5) by step 4) in the centrifugal precipitation obtained add 0.5mol/L acetic acid and suspend and dissolve, it is placed on magnetic stirrer and stirs extraction 24h��26h, it is 0��4 DEG C with High speed refrigerated centrifuge in temperature, centrifugal 10��30min when rotating speed is 6000 turns/min, take second time supernatant solution;
6) merge described first time supernatant solution and described the twice supernatant solution, be slowly added to alkaline solution and regulate pH value to 5, filter precipitate, remaining solution adds solid sodium chloride powder, it is slowly stirred, at 4 DEG C, stands 8��14 hours and form sheep scleral collagen stock solution;
7) sheep scleral collagen stock solution is positioned in High speed refrigerated centrifuge, it it is 0��4 DEG C in temperature, rotating speed is 6000 turns/min, centrifugal when the time is 10��30min, takes precipitation, dissolve with spirit of vinegar, lysate is loaded in bag filter, respectively dialyse 24��26 hours with the acetic acid solution of gradient concentration respectively, then dialyse 24��26 hours with distilled water, within every 24��26 hours, change dialysis solution twice, obtain sheep scleral collagen aqueous solution;
8) by sheep scleral collagen aqueous solution lyophilization at-20 DEG C��-80 DEG C, sheep scleral collagen is prepared.
Step 6) described in alkaline solution be the KOH of NaOH or 6mol/L of 6mol/L.
In the sheep scleral collagen gel for eye use of the present invention, described polyvinyl alcohol is thickening agent, described boric acid, Borax are that ion buffering is right, described sodium chloride is osmotic pressure regulator, described tween is solubilizing agent, described tween is tween 80, and described preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide.
The preparation method that the present invention also provides for a kind of sheep scleral collagen gel for eye use, the method aseptically carries out, and its specific make step is:
1) take boric acid that mass fraction is 0.5%��2% and Borax, mass fraction are the polyvinyl alcohol of 0.5%��2%, mass fraction is the sodium chloride of 0.5%��2%, mass fraction is the tween of 0.3%��2%, the sheep scleral collagen that preservative that mass fraction is 0.01%��2%, mass fraction are 80%��90%, and the water for injection of surplus is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of 75��85 DEG C, add tween, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, on filter, add water for injection, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use.
Described sheep scleral collagen gel for eye use is white gels shape, and pH value is 6.5��7.4.
In the preparation of sheep scleral collagen gel for eye use, the polyvinyl alcohol adopted is thickening agent, described boric acid, Borax are that ion buffering is right, described sodium chloride is osmotic pressure regulator, described tween is solubilizing agent, described tween is tween 80, and described preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide.
Compared with prior art, first, the present invention is extraction collagen protein from the eye of animal sheep, from the collagen protein that sheep eye sclera extracts, except the good characteristic with animal collagen, also have and seemingly spend height with human eye collagen fabric picture, safety, virus-free hidden danger, molecular weight, feature soluble in water, that histocompatibility is good. In the sheep scleral collagen gel for eye use of preparation, employing polyvinyl alcohol is thickening agent, can increase the viscosity of gel, extends the time of contact of medicine and ocular tissue; Boric acid, Borax are that ion buffering is right, keep ocular fluid pH value stable about 7.2; Sodium chloride is osmotic pressure regulator, makes gel close with the osmotic pressure of tissue; Solubilizing agent is tween, and preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide. Secondly, the extracting method of sheep scleral collagen adopts enzyme hydrolysis collagen protein mild condition, the collagen protein purity extracted is high, good water solubility, stable in physicochemical property, the gel for eye use using eye sclera collagen protein to make, can effectively reinforce Posterior pole scleral tissue, suppress axis oculi to extend further, control myopia progression.
Detailed description of the invention
Following example, are further elaborated to the present invention. Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
Fresh sheep eyeball is removed other parts such as vitreous body, retina and the pigmented film etc. beyond sclera, isolates sheep sclera, be cut into sheep sclera thin slice. Take 100g sheep eye sclera fragment, by 9g/L normal saline 1000ml soaking and washing repeatedly, remove serum and fat, then with 1000ml distilled water rinsed clean, dry in an aseptic environment, obtain sheep sclera dry tablet standby. Taking 100g sclera fragment and add 10g pepsin, namely pepsic quality is the 10% of sheep sclera dry tablet quality, homogenate on refiner, is placed on magnetic stirrer and is slowly stirred extraction 24h��26h, stirs 24h in the present embodiment. Then being 0��4 DEG C with High speed refrigerated centrifuge in temperature, when rotating speed is 6000 turns/min, centrifugal 10��30min, takes first time supernatant solution, is precipitated. Precipitation adds 0.5mol/L acetic acid 50ml suspension dissolve, it is placed on magnetic stirrer stirring extraction 24��26h, the present embodiment stirs 24h, be 0��4 DEG C with High speed refrigerated centrifuge in temperature, with the centrifugal 10��30min of 6000 turns/min, take second time supernatant solution. Merge twice supernatant, be slowly added to 6mol/LNaOH (KOH) solution and regulate pH value to 5, filter precipitate, adding 70g solid sodium chloride powder in remaining solution, be slowly stirred, 4 DEG C of standings overnight preserve, time of repose is 8��14 hours, forms sheep scleral collagen stock solution. It is 0��4 DEG C with High speed refrigerated centrifuge in temperature, with the centrifugal 10��30min of 6000 turns/min, take precipitation, again dissolve with spirit of vinegar, lysate is loaded in bag filter, with mass fraction respectively 0.1%, the acetic acid solution of 0.01% respectively dialyses 1 day (24��26 hours), again with 1 day (24��26 hours) of distilled water dialysis, every day, (24��26 hours) changed dialysis solution twice, and what finally obtain is the collagen aqueous solution that purity is higher. Finally, frozen drying method is adopted to prepare into pure collagen protein at-20 DEG C��-80 DEG C the collagen aqueous solution of purification.
The present invention utilizes enzyme hydrolysis-salt extraction method to extract collagen protein from the eye of animal sheep, from the collagen protein that sheep eye sclera extracts, except the good characteristic with animal collagen, also have and seemingly spend height with human eye collagen fabric picture, safety, virus-free hidden danger, molecular weight, feature soluble in water, that histocompatibility is good. Effectively overcome ophthalmically acceptable collagen protein in prior art and there is defect problems such as infecting disease, cause rejection, molecular weight big.
The present invention also provides for a kind of sheep scleral collagen gel for eye use, including sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 80%��90%, the mass fraction of preservative is 0.01%��2%, the mass fraction of boric acid and Borax is 0.5%��2%, the mass fraction of polyvinyl alcohol is 0.5%��2%, the mass fraction of sodium chloride is 0.5��2%, and the mark of tween quality is 0.3%��2%, and the mass fraction of water for injection is surplus.
The preparation method that the present invention also provides for a kind of sheep scleral collagen gel for eye use, the method aseptically carries out, and its specific make step is:
1) take boric acid that mass fraction is 0.5%��2% and Borax, mass fraction are the polyvinyl alcohol of 0.5%��2%, mass fraction is the sodium chloride of 0.5%��2%, mass fraction is the tween of 0.3%��2%, the sheep scleral collagen that preservative that mass fraction is 0.01%��2%, mass fraction are 80%��90%, and the water for injection of surplus is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of 75��85 DEG C, add tween 80, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, add on filter inject water to enough, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use. The sheep scleral collagen gel for eye use prepared is white gels shape, and pH value is 6.5��7.4.
In the preparation of sheep scleral collagen gel for eye use, employing polyvinyl alcohol is thickening agent, can increase the viscosity of gel, extends the time of contact of medicine and ocular tissue; Boric acid, Borax are that ion buffering is right, keep ocular fluid pH value stable about 7.2; Sodium chloride is osmotic pressure regulator, makes gel close with the osmotic pressure of tissue; Tween is solubilizing agent, and tween selects tween 80, and preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide.
The preferred following example of the present invention.
Embodiment 2
A kind of sheep scleral collagen gel for eye use, including sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 88%, the mass fraction of preservative is 0.05%, the mass fraction of boric acid and Borax is 0.5%, the mass fraction of polyvinyl alcohol is 0.5%, the mass fraction of sodium chloride is 0.5%, and the mark of tween quality is 0.5%, and the mass fraction of water for injection is 9.95%. Described sheep scleral collagen extracts from the sclera of sheep eyeball according to the extracting method of the sheep scleral collagen in embodiment 1.
The preparation of sheep scleral collagen gel for eye use, the method aseptically carries out, and its specific make step is:
1) take boric acid that mass fraction is 0.5% and Borax, mass fraction are the polyvinyl alcohol of 0.5%, mass fraction is the sodium chloride of 0.5%, mass fraction is the tween of 0.5%, the sheep scleral collagen that preservative that mass fraction is 0.05%, mass fraction are 88%, and the water for injection that mass fraction is 9.95% is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of about 80 DEG C, add tween 80, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, add on filter inject water to enough, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use. The sheep scleral collagen gel for eye use prepared is white gels shape, and pH value is 7.2.
Embodiment 3
A kind of sheep scleral collagen gel for eye use, including sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 87%, the mass fraction of preservative is 0.08%, the mass fraction of boric acid and Borax is 1%, the mass fraction of polyvinyl alcohol is 1%, the mass fraction of sodium chloride is 1%, and the mark of tween quality is 1%, and the mass fraction of water for injection is 8.92%. Described sheep scleral collagen extracts from the sclera of sheep eyeball according to the extracting method of the sheep scleral collagen in embodiment 1.
The preparation of sheep scleral collagen gel for eye use, the method aseptically carries out, and its specific make step is:
1) take boric acid that mass fraction is 1% and Borax, mass fraction are the polyvinyl alcohol of 1%, mass fraction is the sodium chloride of 1%, mass fraction is the tween of 1%, the sheep scleral collagen that preservative that mass fraction is 0.08%, mass fraction are 87%, and the water for injection that mass fraction is 8.92% is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of about 80 DEG C, add tween 80, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, add on filter inject water to enough, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use. The sheep scleral collagen gel for eye use prepared is white gels shape, and pH value is 7.3.
Embodiment 4
A kind of sheep scleral collagen gel for eye use, including sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 82.5%, the mass fraction of preservative is 0.15%, the mass fraction of boric acid and Borax is 1.5%, the mass fraction of polyvinyl alcohol is 1.5%, the mass fraction of sodium chloride is 1.5%, and the mark of tween quality is 1.5%, and the mass fraction of water for injection is 11.35%. Described sheep scleral collagen extracts from the sclera of sheep eyeball according to the extracting method of the sheep scleral collagen in embodiment 1.
The preparation of sheep scleral collagen gel for eye use, the method aseptically carries out, and its specific make step is:
1) take boric acid that mass fraction is 1.5% and Borax, mass fraction are the polyvinyl alcohol of 1.5%, mass fraction is the sodium chloride of 1.5%, mass fraction is the tween of 1.5%, the sheep scleral collagen that preservative that mass fraction is 0.15%, mass fraction are 82.5%, and the water for injection that mass fraction is 11.35% is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of proper temperature (about 80 DEG C), add tween 80, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, add on filter inject water to enough, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use. The sheep scleral collagen gel for eye use prepared is white gels shape, and pH value is 7.1.
Zoopery
27 healthy Cavia porcelluss, body weight 0.9 �� 0.2Kg, it is randomly divided into A, B, C tri-groups, often group 9, adopt form deprivation mode, undertaken covering 21 days by Cavia porcellus right eye, then every Cavia porcellus right eye is carried out axiallength, diopter, B ultrasonic eye form measurement determine that it has resulted in myopia model. The same day is reached in myopia model, A group is implemented the sheep scleral collagen gel for eye use in the present embodiment 2 and is carried out the outer subconjunctival injection of Cavia porcellus right eye Posterior pole sclera, B group is implemented the normal saline of 0.9% and is carried out the outer subconjunctival injection of Cavia porcellus right eye Posterior pole sclera, and C group blank does not carry out the outer Posterior pole injection of Cavia porcellus right eye sclera. After implementing 1 day, 7 days, 15 days, 30 days, observe the index change of the axiallength of the shielded right eye of Cavia porcellus, diopter, B ultrasonic eye form respectively, and are recorded.
1 day, 7 days, 15 days, 30 days A, B, C group Cavia porcellus right eye diopters, axiallength averages after sheep scleral collagen gel for eye use after table 1 hides 21 days and in the enforcement embodiment of the present invention 2
The data result of table 1 shows:
The axiallength of A group Cavia porcellus is controlled, and myopia degree has no growth, and B ultrasonic eye form has no significant change.
The axiallength of B group Cavia porcellus continues to increase, and myopia degree increases to some extent, the slight protuberance in B ultrasonic eye form Posterior pole local.
The axiallength of C group Cavia porcellus continues to increase, and myopia degree still has growth, B ultrasonic eye form Posterior pole local eminence.
Therefore, this example collagen collagen protein gel for eye use can substantially suppress the growth of axis oculi, has the effect controlling myopia progression preferably.
Described above illustrate and describes the preferred embodiments of the present invention, as previously mentioned, it is to be understood that the present invention is not limited to form disclosed herein, it is not to be taken as the eliminating to other embodiments, and can be used for other combinations various, amendment and environment, and in invention contemplated scope described herein, can be modified by the technology of above-mentioned instruction or association area or knowledge. And the change that those skilled in the art carry out and change are without departing from the spirit and scope of the present invention, then all should in the protection domain of claims of the present invention.
Claims (7)
1. a sheep scleral collagen gel for eye use, it is characterised in that: include sheep scleral collagen, boric acid, Borax, polyvinyl alcohol, sodium chloride, tween, water for injection, preservative; Wherein, the mass fraction of sheep scleral collagen is 80%��90%, the mass fraction of preservative is 0.01%��2%, the mass fraction of boric acid and Borax is 0.5%��2%, the mass fraction of polyvinyl alcohol is 0.5%��2%, the mass fraction of sodium chloride is 0.5��2%, and the mark of tween quality is 0.3%��2%, and the mass fraction of water for injection is surplus.
2. sheep scleral collagen gel for eye use as claimed in claim 1, it is characterised in that the extracting method of described sheep scleral collagen comprises the following steps:
1) in vitro sheep eyeball is removed other parts beyond sclera, isolate sheep sclera;
2) by step 1) in the sheep sclera that obtains be cut into sheep sclera sheet; With normal saline soaking and washing sheep sclera sheet, after removing serum and fat, rinse with distilled water, dry in an aseptic environment, obtain sheep sclera dry tablet;
3) taking described sheep sclera dry tablet, add pepsin, described pepsic quality is the 10% of sheep sclera dry tablet quality, puts into after refiner carries out homogenate, is placed on magnetic stirrer stirring and extracts 24h��26h;
4) being 0��4 DEG C with High speed refrigerated centrifuge in temperature, when rotating speed is 6000 turns/min, centrifugal 10��30min, takes first time supernatant solution, and is precipitated;
5) by step 4) in the centrifugal precipitation obtained add 0.5mol/L acetic acid and suspend and dissolve, it is placed on magnetic stirrer and stirs extraction 24h��26h, it is 0��4 DEG C with High speed refrigerated centrifuge in temperature, centrifugal 10��30min when rotating speed is 6000 turns/min, take second time supernatant solution;
6) merge described first time supernatant solution and described second time supernatant solution, be slowly added to alkaline solution and regulate pH value to 5, filter precipitate, remaining solution adds solid sodium chloride powder, it is slowly stirred, at 4 DEG C, stands 8��14 hours and form sheep scleral collagen stock solution;
7) sheep scleral collagen stock solution is positioned in High speed refrigerated centrifuge, it it is 0��4 DEG C in temperature, rotating speed is 6000 turns/min, centrifugal when the time is 10��30min, takes precipitation, dissolve with spirit of vinegar, lysate is loaded in bag filter, respectively dialyse 24��26 hours with the acetic acid solution of gradient concentration respectively, then dialyse 24��26 hours with distilled water, within every 24��26 hours, change dialysis solution twice, obtain sheep scleral collagen aqueous solution;
8) by sheep scleral collagen aqueous solution lyophilization at-20 DEG C��-80 DEG C, sheep scleral collagen is prepared.
3. sheep scleral collagen gel for eye use as claimed in claim 2, it is characterised in that step 6) described in alkaline solution be the KOH of NaOH or 6mol/L of 6mol/L.
4. sheep scleral collagen gel for eye use as claimed in claim 1, it is characterized in that: described polyvinyl alcohol is thickening agent, described boric acid, Borax are that ion buffering is right, described sodium chloride is osmotic pressure regulator, described tween is solubilizing agent, described tween is tween 80, and described preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide.
5. the preparation method of a sheep scleral collagen gel for eye use as claimed in claim 1, it is characterised in that: the method aseptically carries out, and its making step is:
1) take boric acid that mass fraction is 0.5%��2% and Borax, mass fraction are the polyvinyl alcohol of 0.5%��2%, mass fraction is the sodium chloride of 0.5%��2%, mass fraction is the tween of 0.3%��2%, the sheep scleral collagen that preservative that mass fraction is 0.01%��2%, mass fraction are 80%��90%, and the water for injection of surplus is standby;
2) weigh boric acid, Borax, benzalkonium bromide are dissolved in the water for injection of 75��85 DEG C, add tween, stir evenly;
3) separately take sheep scleral collagen and be dissolved in polyvinyl alcohol, add above-mentioned solution, stirring while adding, mix homogeneously, let cool, with the membrane filtration that microporous filter membrane is 0.22 ��m of aperture, on filter, add water for injection, through 100 DEG C of flowing steam sterilization 30min, aseptic subpackaged make sheep scleral collagen gel for eye use.
6. the preparation method of sheep scleral collagen gel for eye use as claimed in claim 5, it is characterised in that: described sheep scleral collagen gel for eye use is white gels shape, and pH value is 6.5��7.4.
7. the preparation method of sheep scleral collagen gel for eye use as claimed in claim 5, it is characterized in that: described polyvinyl alcohol is thickening agent, described boric acid, Borax are that ion buffering is right, described sodium chloride is osmotic pressure regulator, described tween is solubilizing agent, described tween is tween 80, and described preservative is ethyl hydroxybenzoate, propyl p-hydroxybenzoate or benzalkonium bromide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610076074.2A CN105617350A (en) | 2016-02-03 | 2016-02-03 | Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610076074.2A CN105617350A (en) | 2016-02-03 | 2016-02-03 | Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105617350A true CN105617350A (en) | 2016-06-01 |
Family
ID=56032951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610076074.2A Pending CN105617350A (en) | 2016-02-03 | 2016-02-03 | Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105617350A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113133431A (en) * | 2021-02-25 | 2021-07-20 | 中南大学 | Establishment method, model and application of chronic ocular hypertension combined long-axis animal model |
| CN113633760A (en) * | 2020-04-27 | 2021-11-12 | 北京大学第一医院 | Application of transglutaminase in medicine for inhibiting or delaying myopia |
| CN114634651A (en) * | 2022-05-17 | 2022-06-17 | 天新福（北京）医疗器材股份有限公司 | Sterile porous collagen repair gel and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1591974A1 (en) * | 1988-02-24 | 1990-09-15 | Mezhotraslevoj Nt Kompleks Mik | Method of treating progressive myopia |
| US5094856A (en) * | 1986-10-13 | 1992-03-10 | Moskovsky Nauchno-Teknichesky Complexs "Mikrochirurgija Glaza" | Ophthalmological collagen coverings |
| RU2079296C1 (en) * | 1993-04-29 | 1997-05-20 | Межотраслевой научно-технический комплекс "Микрохирургия глаза" | Method of myopia surgery treatment |
| RU2272599C1 (en) * | 2004-10-05 | 2006-03-27 | Ирина Александровна Маклакова | Biomaterial "kollaplant" for stabilization of progressing myopia |
| CN1895284A (en) * | 2006-06-22 | 2007-01-17 | 武汉远大制药集团有限公司 | Gel for removing ocular by ice pearl and its preparation |
| CN1927391A (en) * | 2005-09-08 | 2007-03-14 | 佟刚 | Collagen eye drops |
-
2016
- 2016-02-03 CN CN201610076074.2A patent/CN105617350A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5094856A (en) * | 1986-10-13 | 1992-03-10 | Moskovsky Nauchno-Teknichesky Complexs "Mikrochirurgija Glaza" | Ophthalmological collagen coverings |
| SU1591974A1 (en) * | 1988-02-24 | 1990-09-15 | Mezhotraslevoj Nt Kompleks Mik | Method of treating progressive myopia |
| RU2079296C1 (en) * | 1993-04-29 | 1997-05-20 | Межотраслевой научно-технический комплекс "Микрохирургия глаза" | Method of myopia surgery treatment |
| RU2272599C1 (en) * | 2004-10-05 | 2006-03-27 | Ирина Александровна Маклакова | Biomaterial "kollaplant" for stabilization of progressing myopia |
| CN1927391A (en) * | 2005-09-08 | 2007-03-14 | 佟刚 | Collagen eye drops |
| CN1895284A (en) * | 2006-06-22 | 2007-01-17 | 武汉远大制药集团有限公司 | Gel for removing ocular by ice pearl and its preparation |
Non-Patent Citations (6)
| Title |
|---|
| E. S. AVETISOV等: "Nonsurgical and surgical methods of sclera reinforcement in progressive myopia", 《ACTA OPHTHALMOLOGICA SCANDINAVICA》 * |
| СИДЕНКО Л. Н等: "Органопрепараты в офтальмологии и ринологии: состояние и перспективы", 《ФАРМАКОМ》 * |
| 孟宪敏等: "猪巩膜胶原的纯化及胶原膜制备的研究", 《中国药学杂志》 * |
| 李二凤等: "胶原蛋白的提取工艺研究", 《食品研究与开发》 * |
| 杨明等: "《药剂学》", 31 August 2014, 中国医药科技出版社 * |
| 陈星星等: "胶原蛋白在眼科疾病方面的研究新进展", 《国际眼科杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113633760A (en) * | 2020-04-27 | 2021-11-12 | 北京大学第一医院 | Application of transglutaminase in medicine for inhibiting or delaying myopia |
| CN113133431A (en) * | 2021-02-25 | 2021-07-20 | 中南大学 | Establishment method, model and application of chronic ocular hypertension combined long-axis animal model |
| CN114634651A (en) * | 2022-05-17 | 2022-06-17 | 天新福（北京）医疗器材股份有限公司 | Sterile porous collagen repair gel and preparation method thereof |
| CN114634651B (en) * | 2022-05-17 | 2022-07-29 | 天新福（北京）医疗器材股份有限公司 | Sterile porous collagen repairing gel and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101018513B (en) | Vision enhancing ophthalmic devices and related methods and compositions | |
| CN109207545B (en) | Collagen extraction method | |
| KR101840726B1 (en) | Method for preparing heterogenous corneal material | |
| US6367480B1 (en) | Methods for visualizing the anterior lens capsule of the human eye | |
| CN113926001B (en) | Bionic cornea and preparation method thereof | |
| CN105617350A (en) | Extraction method of sheep sclera collagen, ophthalmic gel and preparation method thereof | |
| JPH1156897A (en) | Collagen ophthalmologic surgery assisting agent | |
| Andreev et al. | A new collagen scaffold for the improvement of corneal biomechanical properties in a rabbit model | |
| CN109221093A (en) | A kind of cornea tissue long-term preservation liquid and preparation method thereof | |
| CN106420809B (en) | A kind of ophthalmologic operation perfusion liquid | |
| CN107233143A (en) | Remove cell corneal stroma lens and preparation method thereof | |
| CN107233144A (en) | Go application of the cell corneal stroma lens in treatment ophthalmology disease | |
| CN104548201A (en) | Cornea tissue repairing material and preparation method thereof | |
| CN105688282A (en) | Novel biological artificial cornea capable of realizing cellularization through in-vivo induction as well as realizing quick transparency | |
| Han et al. | Corneal stromal filler injection of gelatin-based photocurable hydrogels for maintaining the corneal thickness and reconstruction of corneal stroma | |
| CN113633760A (en) | Application of transglutaminase in medicine for inhibiting or delaying myopia | |
| RU2309749C1 (en) | Method for treating optic nerve atrophy cases | |
| CN108853598A (en) | A kind of preparation of autologous organisms protein hydrogel and its application in operation on retina | |
| Sinha et al. | Visual outcome of cataract surgery with pupillary sphincterotomy in eyes with coexisting corneal opacity | |
| RU2301675C1 (en) | Method for treatment of diabetic retinopathy with preparation "bol-khit" | |
| US20210324025A1 (en) | Compositions and methods for treating corneal endothelium | |
| CN111588736A (en) | Artificial tear and preparation method thereof | |
| RU2281775C1 (en) | Method for treatment of central atherosclerotic chorioretinopathy | |
| Maurice | Clearing media for the eye. | |
| Carrington et al. | The viscous layer overlying the corneal posterior epithelium of the domestic cat. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160601 |