CN102178936A - Methods for preparing collagen oleostock and moistening eyedrops - Google Patents
Methods for preparing collagen oleostock and moistening eyedrops Download PDFInfo
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Abstract
The invention discloses methods for preparing collagen oleostock and moistening eyedrops. The method for preparing collagen oleostok comprises the following steps of: soaking raw materials with bromogeramine; then soaking with the mixed solution of peracetic acid and ethanol for sterilizing and decontaminating; and cleaning the sterilized and decontaminated raw materials and soaking with acetum to obtain the collagen oleostock. The method for preparing moistening eyedrops comprises the following steps of: performing enzymolysis and purification on the collagen oleostock to obtain collagen liquid; adjusting the concentration and osmotic pressure of the collagen liquid, filling under an aseptic condition and finally sealing to obtain disposable moistening collagen eyedrops. By using the method for preparing the collagen oleostock, various microbes and harmful substances in the raw materials can be killed effectively, and collagen cannot be inactivated. The moistening eyedrops prepared by using the method disclosed by the invention has a good treatment effect on dry eye syndrome, can restore cornea, is applicable to eye ball cleaning, lubricating and moistening, routine nursing care on dry eye syndrome and hemostasis and restoration of cornea injury.
Description
Technical field
The present invention relates to the preparation method of a kind of collagen stock solution and profit eye liquid.
Background technology
The cornea and conjunctiva xerosis claims xerophthalmia again, is because the tear film that the unusual or tear hydrodynamics of tear quality and quantity causes unusually is unstable and the infringement of eye table, thereby causes a class disease of ophthalmic uncomfortable symptom.Xerophthalmia is the multifactorial cause of disease, and wherein, it is one of modern's main reason of producing the eye xerophthalmia that life-time service contains the profit eye liquid of antiseptic or conditioning liquid.
Be beneficial to the preparation preservation though add the antiseptic of certain content in the profit eye liquid, life-time service can cause shallow sexual stimulus of normal eye's cornea table or damage, thereby causes or increase the weight of the xerophthalmia symptom.Bromo geramine is widely used in all kinds of profit eye liquid as conventional preservatives, general addition is 0.1g/ml, discover, life-time service contains the profit eye liquid of bromo geramine, can cause irreversible damage to the cornea epithelial cell, visual deterioration appears during beginning, and one's eyes became bloodshot or symptom such as blurred vision, then cause the epithelial cell degeneration, come off, seriously can cause corneal ulcer or erosion.
Collagen twists into spiral fibrous extracellular protein by three peptide chains, and it is proteinic more than 30% to account for whole body, has unique physiologically active.Collagen protein is rich in aminoacid such as the glycine, proline, hydroxyproline of needed by human, it is most important component in the extracellular matrix, have strong biological activity and biological function, can participate in migration, differentiation and the propagation of cell, make bone tendon, cartilage and skin have certain mechanical strength, and because its faint antigenicity and excellent biological compatibility, of many uses at pharmaceutical sanitary fields such as burn, wound, cornea disease, beauty treatment, hard tissue repair, wound surface hemostasis, in ocular tissue, mainly be distributed in cornea and sclera.
Though collagen has biology performance preferably, as a kind of natural activity albumen, if treatment process is improper, very easily degeneration loss of activity can't reach the purpose for the treatment of or keeping healthy in the production.Because collagen is rich in nutrition, be very easy to grow microorganism simultaneously, in this case, generally need in collagen solution, add antiseptic and/or antibiotic, to guarantee that collagen solution does not go bad at lay up period.But add antiseptic and/or antibiotic, can cause side effect such as drug resistance, zest again, severe patient may cause generations such as xerophthalmia, glaucoma.
In ophthalmic preparation, though collagem membrane has been widely used in ophthalmic injuries treatment research, be used for eye daily health caring and care product, there is not sophisticated product to release market at present as yet.But obtain the collagen protein profit eye liquid of practical application, but need a kind of mass preparation aseptic harmless, and can keep the method for the active collagen liquid of collagen.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of collagen stock solution and profit eye liquid.
The technical solution used in the present invention is:
The former liquid and preparation method thereof of a kind of collagen comprises the steps:
1) raw material is used the bromo geramine solution soaking, used peracetic acid and alcohol mixed solution to soak afterwards, carry out disinfection and harmless treatment;
2) will sterilize and the raw material of harmless treatment cleans up, soak 12～48 hours, obtain collagen stock solution with acetum.
Preferably, the concentration of described bromo geramine solution is 0.1～0.5%.
Preferably, the soak time of described bromo geramine solution is 10～25min.
Preferably, in peracetic acid and the alcohol mixed solution, the volumetric concentration of peracetic acid is 0.1～0.3%, and alcoholic acid volumetric concentration is 70～75%.
Preferably, the soak time of described peracetic acid and alcohol mixed solution is 10～30min.
Preferably, the concentration of described acetum is 0.05～1.0M.
A kind of preparation method of disposable collagen profit eye liquid may further comprise the steps:
1) molten, the purification of collagen stock solution acid that said method is made obtains collagen liquid;
2) concentration and the osmotic pressure of adjusting collagen liquid, fill under the aseptic condition, sealing obtain disposable collagen profit eye liquid.
Preferably, the specification of described disposable collagen profit eye liquid is that 0.1～2ml/ props up.
Described disposable collagen profit eye liquid be applicable to eyeball cleaning, lubricatedly preserve moisture, the daily nursing of xerophthalmia and the hemostasis reparation of corneal injury wound surface.
The preparation method of collagen stock solution of the present invention can be killed various microorganisms and harmful substance in the raw material effectively.Simultaneously, can not make the active inactivation of collagen, guarantee that the various collagen products that make have good biological activity.
The profit eye liquid that the inventive method makes; form by collagen protein, osmotic pressure regulator and water for injection; xerophthalmia is had good curative effect, but the effective for repairing cornea, be applicable to eyeball cleaning, lubricated preserve moisture, the daily nursing of xerophthalmia and the hemostasis reparation of corneal injury wound surface.Do not contain antiseptic, antibiotic in the profit eye liquid, avoided the injury of antiseptic, antibiotic effectively eyes.
Description of drawings
Fig. 1 is the pathology figure of model contrast eye in the zoopery;
Fig. 2 is to use the profit eye liquid of the inventive method preparation to be used for the pathology figure of the treatment eye of animal experiment study;
Fig. 3 is the treatment research effect figure that the profit eye liquid of the inventive method preparation is used for the cornea repair in trauma;
Fig. 4 is the ultraviolet absorption peak qualitative detection figure of the profit eye liquid of the inventive method preparation;
Fig. 5 is the high performance liquid chromatography qualitative detection figure of the profit eye liquid of the inventive method preparation;
Fig. 6 is the SDS-PAGE electrophoretogram of the profit eye liquid of the inventive method preparation.
The specific embodiment
The former liquid and preparation method thereof of a kind of collagen comprises the steps:
1) raw material is used the bromo geramine solution soaking, used peracetic acid and alcohol mixed solution to soak afterwards, carry out disinfection and harmless treatment;
2) will sterilize and the raw material of harmless treatment cleans up, soak 12～48 hours, obtain collagen stock solution with acetum.
Preferably, the concentration of described bromo geramine solution is 0.1～0.5%.
Preferably, the soak time of described bromo geramine solution is 10～25min.
Preferably, in peracetic acid and the alcohol mixed solution, the volumetric concentration of peracetic acid is 0.1～0.3%, and alcoholic acid volumetric concentration is 70～75%.
Preferably, the soak time of described peracetic acid and alcohol mixed solution is 10～30min, and is better, and soak time is 10～20min.
Preferably, the concentration of described acetum is 0.05～1.0M, is preferably 0.05～0.2M.The soak time of acetum is preferably 36～48 hours.
A kind of preparation method of disposable collagen profit eye liquid may further comprise the steps:
1) molten, the purification of collagen stock solution acid that said method is made obtains collagen liquid;
2) concentration and the osmotic pressure of adjusting collagen liquid, fill under the aseptic condition, sealing obtain disposable collagen profit eye liquid.
Preferably, the specification of described disposable collagen profit eye liquid is that 0.1～2ml/ props up, and makes things convenient for the use of different crowd, avoids waste.
Described disposable collagen profit eye liquid be applicable to eyeball cleaning, lubricatedly preserve moisture, the daily nursing of xerophthalmia and the hemostasis reparation of corneal injury wound surface.
The raw material of preparation collagen protein is well-known to those skilled in the art, and commonly used have fresh Mus tail, pig, a tendon Bovis seu Bubali tendon etc.Certainly, according to those skilled in the art's basic general knowledge, before the harmless treatment that carries out disinfection, need earlier these raw materials to be cleaned, to remove unwanted fur, serum, fat and other spots.
In order to guarantee the treatment effect of raw material, preferably under cleaning condition, handle, as in the harmless treatment that carries out disinfection under ten thousand grades of conditions of GMP or under the more clean environment.Certainly, according to those skilled in the art's basic general knowledge, for protection profit eye liquid is aseptic, fill, encapsulation process should be carried out under the condition of local laminar flow.
Below in conjunction with embodiment, further specify the present invention.
Usually material, Mus tails such as the muscle tendon of employing cattle or pig, skin, bone carry out the extraction and the preparation of I Collagen Type VI.Before the extraction, raw material extracts with acetic acid after carrying out corresponding unhairing, degrease processing and thorough the cleaning.The densification of above-mentioned material quality, contain solubility and insoluble collagen.
Collagen tissue is loosened, is soluble collagen in the fresh Mus tail, and the stock solution homogeneity of extracting after preparing is good, but the collagen that extracts is equally easily destroyed by chemical reagent.No matter adopt which kind of material preparation collagen, the sterilization of raw material and the method for harmless treatment are very big to the quality influence of the collagen of extraction.
In following examples, be example, illustrate how to prepare collagen stock solution and profit eye liquid with fresh Mus tail.
In following examples, described percentage concentration, except that peracetic acid, alcoholic acid percentage concentration are the volumetric concentration, other all refer to mass percentage concentration.
The preparation of collagen stock solution
1) fresh Mus tail water is cleaned up, remove spot, serum on serum deprivation and the fur;
2) the Mus tail that cleans up is soaked 15min in 0.5% bromo geramine solution, unite 75% ethanol in 0.3% peracetic acid after cleaning and soak 10min;
3) with Mus tail decortication extracting muscle tendon, clean up, soak to extract in 0.05M acetic acid low temperature and handle 48h, obtain collagen stock solution.
Embodiment 2
1) fresh Mus tail water is cleaned up, remove spot, serum on serum deprivation and the fur;
2) the Mus tail that cleans up is soaked 25min in 0.1% bromo geramine solution, unite 70% ethanol in 0.1% peracetic acid after cleaning and soak 20min;
3) with Mus tail decortication extracting muscle tendon, clean up, extract in 0.1M acetic acid soak at room temperature and handle 36h, obtain collagen stock solution.
Embodiment 3
1) fresh Mus tail water is cleaned up, remove spot, serum on serum deprivation and the fur;
2) the Mus tail that cleans up is soaked 20min in 0.2% bromo geramine solution, unite 70% ethanol in 0.3% peracetic acid after cleaning and soak 30min;
3) with Mus tail decortication extracting muscle tendon, clean up, extract in 0.3M acetic acid soak at room temperature and handle 24h, obtain collagen stock solution.
Embodiment 4
1) fresh Mus tail water is cleaned up, remove spot, serum on serum deprivation and the fur;
2) the Mus tail that cleans up is soaked 15min in 0.3% bromo geramine solution, unite 75% ethanol in 0.2% peracetic acid after cleaning and soak 10min;
3) with Mus tail decortication extracting muscle tendon, clean up, extract in 0.5M acetic acid soak at room temperature and handle 30h, obtain collagen stock solution.
1) fresh Mus tail water is cleaned up, remove spot, serum on serum deprivation and the fur;
2) the Mus tail that cleans up is soaked 10min in 0.4% bromo geramine solution, unite 75% ethanol in 0.3% peracetic acid after cleaning and soak 12min;
3) with Mus tail decortication extracting muscle tendon, clean up, extract in 1M acetic acid soak at room temperature and handle 12h, obtain collagen stock solution.
The collagen stock solution that the foregoing description is made according to 2010 editions the 3rd sterile detection methods of the Chinese Pharmacopoeia detection of taking a sample, the result shows qualified, shows that collagen stock solution is aseptic.
The preparation of profit eye liquid
Profit eye liquid A
1) get the collagen stock solution that embodiment 1 prepares, saltout according to a conventional method, dialyse under hundred grades of laminar flow hood, purification obtains collagen liquid;
2) concentration of using glucose solution and water for injection to regulate collagen liquid is 0.5mg/ml, and osmotic pressure oozes for waiting;
3) under hundred grades of conditions, adopt and blow the embedding integration apparatus and carry out 0.8ml/ and prop up fill, seal, one-body molded, obtain disposable collagen profit eye liquid.
Profit eye liquid B
1) get the collagen stock solution that embodiment 2 prepares, saltout according to a conventional method, dialyse under hundred grades of laminar flow hood, purification obtains collagen liquid;
2) concentration of using glucose solution and water for injection to regulate collagen liquid is 3mg/ml, and osmotic pressure oozes for waiting;
3) under hundred grades of conditions, adopt and blow the embedding integration apparatus and carry out 2.0ml/ and prop up fill, seal, one-body molded, obtain disposable collagen profit eye liquid.
Clinical trial
Extract the lachrymal gland of lagophthalmos, merge and use 0.1% bromo geramine solution to carry out the xerophthalmia modeling.
The treatment group is moistened eye liquid A treatment, and 3 times/d, model group gives normal saline, continuously 14d; Eye is apparent to be examined and brave very popular actor or actress film dyeing, eyelid tissue pathology checking.
The result: treatment group bloodshot eyes, edema symptom are subjected to the examination eye light than model group, and the red painted score value of tiger is compared all carrying out property minimizings with matched group.
The pathology figure of matched group as shown in Figure 1, the pathology figure of treatment group is as shown in Figure 2.As can be seen from the figure, in the matched group, vasodilation hyperemia under the eyelid epithelia, glandular hyperplasia in the tarsus; In the treatment group, eyelid epithelia is the columnar epithelium that contains goblet cell, and last veins beneath the skin does not see obvious expansion, and body of gland is not seen obvious hypertrophy in the tarsus, and a matter is not seen obvious cell infiltration.Treatment group rabbit cornea epithelial cell level increases, and endodermis cell vacuolar degeneration is few, and eyelid organizes under the epithelium in congested degree of vasodilation and the tarsus body of gland hyperplasia degree all light than matched group.
Above-mentioned experimental result shows, the profit eye liquid of the inventive method preparation can be by lubricated to preserving moisture of eyeball and promote near the eyes approach such as regeneration and restoration that xerophthalmia is played certain alleviation and improvement effect.
Test 2
Model: rabbit corneal mechanical damage model;
With indexs such as conjunctival secretion and edema degree, the scoring of 2% fluorescein corneal dyeing, wound healing rates, estimate the repair of profit eye liquid B to the damage cornea.
The result: matched group drips normal saline, and conjunctival secretion increases behind all animal surgeries of matched group, and edema is serious, and the 3rd day corneal wound healing rate is the 39.7 ± 26.1%, 13rd day healing rate 85.5 ± 14.1%; Treatment is organized all animal postoperatives and is used collagen profit eye liquid, and conjunctival secretion is less, no serious edema and hyperemia, and the 3rd day corneal wound healing rate is that the 42.4 ± 23.2%, 13rd day healing rate is 100%.From the 9th day, experimental group and matched group corneal wound healing had significant difference (P＜0.05), as Fig. 3.Left figure among Fig. 3 is the corneal injury situation that matched group used behind the normal saline the 14th day; Right figure is that corneal injury was healed substantially after experimental group used collagen profit eye liquid 14.The result shows: compare with matched group, the corneal wound healing rate that the treatment group was put in each observing time all is higher than matched group, this profit eye liquid can be accelerated the repair of rabbit damage cornea, and the prompting said preparation can be applied to clinical operation on cornea patient's postoperative reparation and nursing.
The evaluation of collagen
The collagen that Sigma company produces is the purity height, and stable performance is used as standard collagen at present.
Get a profit eye liquid A and carry out the ultraviolet absorption peak qualitative detection, testing result as shown in Figure 4, as seen from the figure, the collagen in the profit eye liquid has a characteristic absorption peak at ultraviolet 225nm place, and is suitable with Sigma company reference substance.
Get profit eye liquid A and carry out the high performance liquid chromatography qualitative detection, testing result as seen from the figure, at retention time 2～3s place one characteristic peak is arranged, and purity is up to 99.9% as shown in Figure 5.
Get the collagen that profit eye liquid A and Sigma company produce and carry out the SDS-PAGE electrophoresis, electrophoretogram as shown in Figure 6, as seen from the figure, contained collagen has distinctive 4～6 bands in the profit eye liquid, and is suitable with the Sigma reference substance.
More than test as can be known, the collagen profit eye liquid that uses collagen stock solution of the present invention to produce to obtain, collagen purity height has wherein kept the biological activity of collagen.
Claims (9)
1. the former liquid and preparation method thereof of collagen comprises the steps:
1) raw material is used the bromo geramine solution soaking, used peracetic acid and alcohol mixed solution to soak afterwards, carry out disinfection and harmless treatment;
2) will sterilize and the raw material of harmless treatment cleans up, soak 12～48 hours, obtain collagen stock solution with acetum.
2. the former liquid and preparation method thereof of a kind of collagen according to claim 1 is characterized in that: the concentration of described bromo geramine solution is 0.1～0.5%.
3. the former liquid and preparation method thereof of a kind of collagen according to claim 1 and 2 is characterized in that: the soak time of described bromo geramine solution is 10～25min.
4. the former liquid and preparation method thereof of a kind of collagen according to claim 1 is characterized in that: in described peracetic acid and the alcohol mixed solution, the volumetric concentration of peracetic acid is 0.1～0.3%, and alcoholic acid volumetric concentration is 70～75%.
5. according to claim 1 or the former liquid and preparation method thereof of 4 described a kind of collagens, it is characterized in that: the soak time of described peracetic acid and alcohol mixed solution is 10～30min.
6. the former liquid and preparation method thereof of a kind of collagen according to claim 1 is characterized in that: the concentration of described acetum is 0.05～1.0M.
7. the preparation method of a disposable collagen profit eye liquid may further comprise the steps:
1) molten, the purification of collagen stock solution acid that any described method of claim 1～6 is made obtains collagen liquid;
2) concentration and the osmotic pressure of adjusting collagen liquid, fill under the aseptic condition, sealing obtain disposable collagen profit eye liquid.
8. according to the preparation method of the described a kind of disposable collagen profit eye liquid of claim 7, it is characterized in that: the specification of described disposable collagen profit eye liquid is that 0.1～2ml/ props up.
9. according to the preparation method of the described a kind of collagen profit of claim 7 eye liquid, it is characterized in that: described disposable collagen profit eye liquid be applicable to eyeball cleaning, lubricatedly preserve moisture, the daily nursing of xerophthalmia and the hemostasis reparation of corneal injury wound surface.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104721877A (en) * | 2015-03-18 | 2015-06-24 | 广州创尔生物技术股份有限公司 | Sterile collagen dressing and preparation method thereof |
| CN109260458A (en) * | 2017-07-18 | 2019-01-25 | 百捷生物科技有限公司 | Collagen Implant is preparing the purposes in operation for glaucoma auxiliary reconstruction |
| CN113713086A (en) * | 2021-08-20 | 2021-11-30 | 山西锦波生物医药股份有限公司 | Composition containing recombinant III-type humanized collagen and preparation method and application thereof |
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| WO2010078883A2 (en) * | 2008-12-18 | 2010-07-15 | Bode Chemie Gmbh | Combined disinfection and decontamination agent having increased effectivity |
| CN101874903A (en) * | 2009-11-20 | 2010-11-03 | 胡庆柳 | Method for preparing collagen artificial skin |
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2011
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| CN1080872A (en) * | 1993-04-15 | 1994-01-19 | 广州市红十字会医院 | Cornea for external use and collagen solution for burn and preparation technology thereof |
| WO2010078883A2 (en) * | 2008-12-18 | 2010-07-15 | Bode Chemie Gmbh | Combined disinfection and decontamination agent having increased effectivity |
| CN101874903A (en) * | 2009-11-20 | 2010-11-03 | 胡庆柳 | Method for preparing collagen artificial skin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104721877A (en) * | 2015-03-18 | 2015-06-24 | 广州创尔生物技术股份有限公司 | Sterile collagen dressing and preparation method thereof |
| CN104721877B (en) * | 2015-03-18 | 2017-09-22 | 广州创尔生物技术股份有限公司 | A kind of sterile collagen sticking dressing and preparation method thereof |
| CN109260458A (en) * | 2017-07-18 | 2019-01-25 | 百捷生物科技有限公司 | Collagen Implant is preparing the purposes in operation for glaucoma auxiliary reconstruction |
| CN109260458B (en) * | 2017-07-18 | 2022-03-18 | 百捷生物科技有限公司 | Application of collagen implant in preparation of glaucoma surgery auxiliary restoration material |
| CN113713086A (en) * | 2021-08-20 | 2021-11-30 | 山西锦波生物医药股份有限公司 | Composition containing recombinant III-type humanized collagen and preparation method and application thereof |
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| CN102178936B (en) | 2013-06-05 |
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Address after: 510663, No. four, No. 1, building A, 17 incense Hill Road, Guangzhou hi tech Industrial Development Zone, Guangdong, China Patentee after: Guangzhou Trauer Biotechnology Co., Ltd. Address before: 510663, No. four, No. 1, building A, 17 incense Hill Road, Guangzhou hi tech Industrial Development Zone, Guangdong, China Patentee before: Guangzhou Trauer Biotechnology Co., Ltd. |
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Inventor after: Tong Gang Inventor after: Lei Jing Inventor after: Chen Danchang Inventor after: Lin Yuquan Inventor after: Zhao Peng Inventor after: Huang Dongjing Inventor after: Huang Sheng Inventor before: Lei Jing Inventor before: Chen Danchang Inventor before: Lin Yuquan Inventor before: Zhao Peng Inventor before: Huang Dongjing Inventor before: Tong Gang Inventor before: Huang Sheng |
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