CN103182075B - Lysozyme preparation, preparation method thereof, and application thereof - Google Patents
Lysozyme preparation, preparation method thereof, and application thereof Download PDFInfo
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- CN103182075B CN103182075B CN201110453880.4A CN201110453880A CN103182075B CN 103182075 B CN103182075 B CN 103182075B CN 201110453880 A CN201110453880 A CN 201110453880A CN 103182075 B CN103182075 B CN 103182075B
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Abstract
The invention belongs to the fields of enzymology and pharmaceutics, and relates to a lysozyme preparation, a preparation method thereof, and an application thereof. Specifically, the lysozyme preparation is prepared through the steps that: (1) lysozyme is dissolved in injection water, such that a lysozyme solution is obtained; (2) a stabilizing agent salt is added into the lysozyme solution obtained in the step (1); and (3) the pH is regulated to 4.6-9.0. The lysozyme preparation provide by the invention can be used in ophthalmic preparations, and can maintain good stability.
Description
Technical field
The invention belongs to zymetology and pharmaceutics field, relate to a kind of lysozyme formulation, Preparation Method And The Use.
Background technology
In recent years, clinical data and the research data of a large amount of ophthalmic pharmaceutical formulation show, the side effect produced in the therapeutic process of ocular disease or untoward reaction have many times all because antibacterial causes.Such as, when using medicine to carry out aftertreatment after operated eye, the corneal epithelial cell caused due to the impact of the antibacterial phenomenon such as to come off happens occasionally.Further, along with popularizing of computer, air-conditioning etc., patients with dry eye crowd is also in continuous increase, and the treatment of this kind of patient often needs to be aided with artificial tears for a long time, and the life-time service of chemical antibacterial can cause corneal injury in various degree.
Therefore, the side effect that the antibacterial in ophthalmic preparation brings has become social question of common concern, is also the focus of current global ophthalmology drug development.The injury that multiple method is brought to avoid antibacterial eye is adopted, as made the ophthalmology single dosage products of not bacteriostatic agent: as the Levofloxacin eye drop of Echinopanax japonicum sky company in prior art; Also degerming ophthalmic preparation container can be used as the UF-021 eye drop of Japanese drop institute exploitation.But the improvement of these packaging aspects can not ensure integral asepsis, and cost is high, for the crowd of life-time service, the financial burden of patient can be increased undoubtedly.
Also someone attempted not adding antibacterial in quinolones eye drop, directly relied on the antibacterial action of medicine to maintain the aseptic condition of preparation.But due to the antimicrobial spectrum of quinolones and inhibitory effect limited, its fungistatic effect is also desirable not to the utmost, does not possess general applicability.
The enzyme of lysozyme (Lysozyme) to be molecular weight be 14.4kDa, nineteen twenty-two Alexander Fleming has found lysozyme in the antibiotic process of searching.As one of the infection natural cover for defense of human body self, it can by 1 between-acetylmuramic acid and 2-Acetamido-2-deoxy-D-glucose residue and in chitodextrin between N-acetyl-glucosamine residue in catalysis Peptidoglycan, the hydrolysis of 4-β chain, and destroy the cell wall of antibacterial, reach the object of killing antibacterial, therefore as antibacterial, can be applied in food sectors.Lysozyme is as enzyme tool high efficiency, and its antibacterial activity is tens times even thousands of times of similar chemical substance; And as human body intrinsic activity material, without any side effects to human body, can be applicable to all kinds of crowd.In addition, lysozyme is with low cost, obtain by way of many, originate wide, in Ovum Gallus domesticus album, milk, all contain a large amount of lysozyme, particularly in recent years along with the extensive use of clone technology, making microorganism extract lysozyme also becomes the large approach of lysozyme mass-produced.
But the less stable of lysozyme in water, can not preserve for a long time.And ophthalmic preparation is water soluble preparation mostly, therefore lysozyme is applied to ophthalmic preparation and is subject to a definite limitation.
Summary of the invention
The present inventor, through deep research and performing creative labour, obtains a kind of lysozyme formulation.The present inventor is surprised to find, and this lysozyme formulation can be used in ophthalmic preparation, and lysozyme can keep good stability in system of the present invention (comprising suitable pH and stabilizing agent salt density).Thus provide following invention:
The comprehensive above problem of present inventor, search out a kind of to eye safety, non-stimulated and efficient antibacterial---lysozyme, this antibacterial is applicable to all kinds of crowd, and with low cost.Lysozyme is 0.001%-25% (w/w) as a kind of antibacterial amount ranges of ophthalmic preparation, and preferable range is 0.01%-20% (w/w), is more preferably 0.1%-15% (w/w); More preferably 0.3%-10% (w/w), is particularly preferably 0.5%-5% (w/w).And with other antibacterial conbined usage, the concentration of two kinds of antibacterial can be made to reduce simultaneously, consumption reduce, reduce the infringement of other antibacterial to eye table.
Though the past has people to consider antibacterial using it as ophthalmic preparation, because its stability is in aqueous not good, lysozyme application is in this regard made to fail to be developed.For ensureing the stability of lysozyme, inventor is by a large amount of experiments, and process lysozyme raw material, makes the refining lysozyme of acquisition that 3-5 can be kept in aqueous to stablize, captured this difficult problem.Treatment process is as follows:
1) lysozyme is dissolved in water for injection, obtains lysozyme soln;
2) to step 1) in lysozyme soln in add stabilizing agent salt;
3) pH to 4.6-9.0 is regulated.
In one embodiment of the invention, also comprise step 4) leave standstill, make lysozyme fully contact rear filtration with stabilizing agent salt;
In one embodiment of the invention, also comprise 5) add adsorbability material the impurity in solution is adsorbed, obtain the solution of clear, cross after filtering adsorbability material for subsequent use after absorption.
In one embodiment of the invention, also comprising 6) solution that obtains can adopt the mode such as lyophilization, spraying dry, and make it preserve in solid form and use in order to preparation; Also after being adjustable to isoelectric point, IP, collect solid, adopt the modes such as vacuum drying, collect solid; The supplementary materials such as other drug, osmotic pressure regulator, moisturizing thickening agent can also be added directly feed intake and make ophthalmic preparation, the dosage forms such as such as eye drop, Eye ointments, gel for eye, eye solid preparation by liquid form.
Here is the detailed explanation to above-mentioned technique:
The dosage form of ophthalmic preparation involved in invention can be liquid, semisolid, solid dosage forms that the ophthalmology such as eye drop, Eye ointments, gel for eye, eye solid preparation are conventional.
PH value regulator involved in invention, to regulate the pH value of water for injection, now use the acid-base modifier that material can be commonly used for dilute hydrochloric acid, acetic acid, sodium hydroxide etc., can also be that some have the salt of acid-base value regulatory function, as citric acid, boric acid, sodium dihydrogen phosphate, sodium hydrogen phosphate etc.
It will be appreciated by those skilled in the art that pH value is very important technical controlling index in ophthalmic preparation, be related to the stability of ophthalmic preparation of the present invention, effectiveness and eye irritation.In addition, pH value is also an important Con trolling index for protein, and as everyone knows, protein has a characteristic index---isoelectric point, IP.The pH value of water solution of protein from isoelectric point, IP more close to, its stability is in aqueous poorer, far away then more stable on the contrary.And easily there is irreversible reaction of degeneration in strong acid and strong base in protein, namely our usually said protein denaturization.Lysozyme, as a kind of enzyme of protide, has the common property of protein equally.
The present invention is the ophthalmic preparation containing lysozyme, needs the feature simultaneously taking into account lysozyme and ophthalmic preparation in preparation process, and therefore the control of pH value can be described as the key in key in this product.
In the process of lysozyme purification in the past, pH value is all below 4.6, and its main cause is ensureing, under the prerequisite that lysozyme is stable, to improve the yield of lysozyme.But inventor finds, under this pH value condition, the lysozyme formulation obtained is excessively strong to people's Eye irritation, but after being increased by pH value, its stability reduces again.By a large amount of experiments, the present inventor finds that solution ph, salinity and process lysozyme concentration are most important in lysozyme Feedstock treating process, can realize with under the pH value of human body proximity by these three parameter values of precise hard_drawn tuhes, obtain more stable lysozyme soln, and lysozyme can be obtained higher yield in processing procedure.By series of experiments, the pH value in the ophthalmic preparation processing procedure of lysozyme is controlled the stability being conducive to improving lysozyme between 4.6-10.0 by inventor, is preferably 4.7-9.0; Be more preferably 4.8-8.5; Be particularly preferably 5.0-8.0; More preferably 5.0-7.0; Further be preferably 5.5-6.5, such as 5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4 or 6.5.
Stabilizing agent involved in the present invention, mostly be salt, include but not limited to one or more salts in sodium carbonate, sodium bicarbonate, sodium citrate, boric acid, Borax, potassium dihydrogen phosphate, potassium chloride, calcium chloride etc., be preferably selected from boric acid, Borax, sodium citrate and dipotassium hydrogen phosphate any one or multiple.Be not limited to theoretical restriction, boric acid, Borax, sodium citrate and dipotassium hydrogen phosphate have the effect of pH regulator concurrently simultaneously.Be not limited to theoretical restriction, lysozyme may produce with some salt actions and precipitate, or the space conformation of lysozyme changes, thus causes the change of lysozyme activity and/or stability.Be not limited to theoretical restriction, aforementioned stable agent salt has two kinds of effects concurrently: one is for regulating solution osmotic pressure, and guarantee ophthalmic preparation is identical with the osmotic pressure of tear and reduce Eye irritation.Another act as, and the molecule that the existence of salt can order about same sex electric charge in the configuration of lysozyme keeps stable in the solution with repulsion, and the molecule of the charges of different polarity then can mutually be assembled and precipitate, and is separated from solution.The existence of salt effectively can remove the unstable factor in solution, thus ensures lysozyme stability.The present invention obtains the above mechanism of action of stabilizing agent by lot of experiments, break the simple limitation of the salts such as the sodium chloride in the past generally used, obtain the stabilizing agent salt being more suitable for lysozyme, and add the kind of stabilizing agent salt, make preparing on prescription of preparation more diversified.
Inventor, when contrasting process with untreated lysozyme raw material active, is surprised to find, stabilizing agent salt add the activity that can keep or strengthen lysozyme raw material.Some salt even can be directly used in the pH value regulating solution, buffer salt in prepared preparation after can be used as.
Mention in the present invention as stated above, this product is processed lysozyme concentrated solution by control ph, stabiliser salt concentration, lysozyme concentration three indexs, and obtain the stable ophthalmic preparation containing lysozyme.Therefore in the present invention, the consumption of salt is also one of very important Con trolling index.The amount of salt is usually between 0.001%-30% (w/w); Be preferably 0.01%-25% (w/w), be more preferably 0.1%-20% (w/w); Be particularly preferably 1%-20% (w/w), more preferably 3%-15% (w/w), such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% (w/w).
In foregoing invention involved standing be for ensureing that, under specific pH value, salts substances and lysozyme fully contact and design, contact is fully conducive to better precipitating the charges of different polarity, and eliminating unstability factor, makes the lysozyme of acquisition more stable.Leave standstill usually under 0 DEG C of-40 DEG C of environment (such as room temperature or room temperature) carry out, the time left standstill for being not less than 2 hours, such as 2-48 hour.
The present inventor finds under study for action, can ensure the stability of lysozyme at certain hour though saltout, but in long storage process, the eye drop of lysozyme still there will be a small amount of precipitate.Carry out lot of experiments through inventor, find the process adding adsorption and sedimentation on the basis of above-mentioned salting-out process, more stable being present in solution system of lysozyme can be made.
Adsorbability material involved in foregoing invention, object is to ensure that the lysozyme in liquid exists with the most stable configuration or space conformation.Be not limited to theoretical restriction as protein, lysozyme can exist with various configuration in water, and can change mutually between various configuration.People was once had to study its various configuration, discovery lysozyme in aqueous major part is all that surface is electronegative, and its positively charged is a small amount of, molecule just because of this positive electricity configuration has certain captivation to the molecule with the charges of different polarity (negative electricity), thus causes protein aggregation.Therefore, present inventor adopts some with the adsorbent of negative electricity, adsorbs the unstable factor in solution, increases to make its stability of solution.The adsorbent adopted in the present invention comprises active carbon, kieselguhr, bentonite etc.Use amount is between 0.001%-10% (w/w); Be preferably 0.01-8%; Be more preferably 0.1%-6%.The temperature of absorption is 0 DEG C-80 DEG C; Be preferably 5 DEG C-75 DEG C, 10 DEG C-70 DEG C, 20 DEG C-65 DEG C or 30 DEG C-60 DEG C.Adsorption time is generally 5min-48h; Be preferably 10min-36h, 15min-24h, 20min-12h, 25min-6h or 30min-2h.
Twice filtration is related in treatment process, this filtration can be the simple filtration of monolayer filter paper, filter membrane, can also be in-depth filtration, as sand stick, quartz sand plate, titanium rod, for in-depth filtration cardboard and adapt to the filter type such as filter element filtering of suitability for industrialized production.
Lysozyme after process, can directly feed intake in liquid form; Also can by dry technologies such as lyophilizations, or be adjusted near lysozyme isoelectric point, IP by materials such as alkaline aqueous solution such as sodium hydroxide by its pH value, after making it separate out in a large number, filtration drying, uses with solid state.Above two states all can directly use as antibacterial.
Solution is obtained as adopted freeze drying process process, its lyophilisation condition: by after process to solution put into cryogenic refrigerator and carry out pre-freeze, the temperature of pre-freeze is between-80 DEG C ~-70 DEG C, after usual pre-freeze 8-12 hour, take out and put into rapidly the cold-trap of having lowered the temperature, the initial temperature of cold-trap is between-50 DEG C ~-30 DEG C, after evacuation 1h, after being slowly warming up to-25 DEG C ~-20 DEG C, continuing evacuation 10h, after moisture distils substantially completely, proceed to secondary lyophilizing, in secondary freeze-drying process, after temperature being risen to 10 DEG C, insulation about 5h, until secondary lyophilizing terminates.
As adopted spray drying condition: charging rate is usually at 1.0-3.0ml/min, and inlet air flow temperature is generally 105 DEG C-120 DEG C, and exit flow temperature is at 60 DEG C-80 DEG C, and sprinkler pressure is at 1.0-1.2kg/cm
2.
Also the alkaline solutions such as sodium hydrate aqueous solution can be adopted to regulate its pH to isoelectric point, IP, after collecting solid, adopt 25 DEG C-40 DEG C, vacuum drying about 24h, to its bone dry, collect the pressed powder obtained.
The invention still further relates to any one in the item of following (1)-(12):
1. a preparation method for lysozyme formulation, comprises the steps:
1) lysozyme is dissolved in water for injection, obtains lysozyme soln;
2) to step 1) in lysozyme soln in add stabilizing agent salt;
3) pH to 4.6-9.0 is regulated.
2. the method according to the 1st, wherein, in described lysozyme soln, the concentration of lysozyme is 0.001%-50% (w/w), 0.01%-20% (w/w), 0.1%-15% (w/w), 0.3%-10% (w/w) or 0.5%-5% (w/w).
3. the method according to the 1st, wherein, described stabilizing agent salt be selected from sodium carbonate, sodium bicarbonate, sodium citrate, boric acid, Borax, potassium dihydrogen phosphate, potassium chloride and calcium chloride one or more; One or more preferably in boric acid, Borax, sodium citrate and dipotassium hydrogen phosphate.
4. the method according to the 1st, wherein, described pH is 4.7-9.0,4.8-8.5,5.0-8.0,5.0-7.0 or 5.5-6.5.
5. the method according to the 1st, it also comprises the steps:
4) leave standstill, make lysozyme fully contact rear filtration with stabilizing agent salt, obtain filtrate; Preferably, described in rest on 0 DEG C of-40 DEG C of environment under carry out, time of repose is between 2-48 hour.
6. the method according to the 1st, it also comprises the steps:
5) add electronegative adsorbent to adsorb the impurity in filtrate, obtain the solution of clear, then cross and filter adsorbability material; Preferably, described adsorbent is selected from one or more in active carbon, kieselguhr and bentonite.
7. the method according to the 1st, wherein, the use amount of described adsorbent is 0.001%-10% (w/w), and adsorption time is 5min-48h.
8. the method according to any one of the 1 to 7, it also comprises the steps:
Lysozyme formulation solution is made lysozyme formulation pressed powder; Particularly, lyophilization or spraying dry is used to obtain lysozyme formulation pressed powder.
9. the lysozyme formulation that the method according to any one of the 1 to 8 is obtained.
10. the lysozyme formulation described in the 9th is preparing the purposes in ophthalmic preparation; Particularly, described ophthalmic preparation is eye drop, Eye ointments, gel for eye or eye solid preparation.
11. purposes according to the 10th, wherein, the content of the lysozyme in described ophthalmic preparation is 0.001%-25% (w/w), 0.01%-20% (w/w), 0.1%-15% (w/w), 0.3%-10% (w/w) or 0.5%-5% (w/w).
Lysozyme formulation described in 12. the 9th is as the purposes of antibacterial and/or antiseptic; Particularly, described purposes is the antibacterial of lysozyme formulation as ophthalmic preparation described in the 9th and/or the purposes of antiseptic.
The beneficial effect of the invention
Lysozyme by human body itself intrinsic, as a kind of natural antimicrobial substance resisting the invasion and attack of the harmful substance such as extraneous antibacterial in tear, therefore, to human body without any harm, can ensure when life-time service to eye table not damaged, be a kind of desirable antibacterial.And as natural bacteriostatic agent, the increase of dosage can not produce any side effect to human body, therefore can suitable increased dosage amount in order to strengthen inhibitory effect, make the ophthalmic preparation of acquisition while reaching good stability, ensure absolute safety.And form compound preservative with chemical antibacterial, chemical antibacterial consumption can be made to be reduced to the consumption less to human body infringement, and not reduce overall antiseptic effect.
Inventor solves lysozyme stability problem in the solution, thus extends the scope of application of lysozyme as antibacterial.First lysozyme is applied in ophthalmic pharmaceutical formulation as ophthalmology antibacterial at home and even in global range.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
embodiment 1: the preparation of lysozyme formulation
(1) getting 0.5g lysozyme raw material adds in water for injection 100g, stirring and dissolving, for subsequent use after all dissolving;
(2) add sodium dihydrogen phosphate 16g wherein, use 1mol/L sodium hydroxide to regulate pH value to 5.0 ± 0.2 of water for injection;
(3) 20 DEG C of standing 48h, cellulose mixture membrane filtration;
(4) add active carbon 0.02g, 70 DEG C of absorption 20min, filtered on buchner funnel removing active carbon, by the solution obtained.
(5) by process after to solution put into cryogenic refrigerator,-70 DEG C of pre-freezes, after usual pre-freeze 8h, take out and put into rapidly the cold well of having lowered the temperature, the initial temperature of cold well is between-40 DEG C, after evacuation 1h, after being slowly warming up to-20 DEG C, continue evacuation 10h, secondary lyophilizing is proceeded to after moisture distils substantially completely, after temperature being risen to 10 DEG C, insulation about 5h, obtains lysozyme formulation after lyophilization.
embodiment 2: the preparation of lysozyme formulation
(1) get boric acid 6.2g, Borax 0.029g adds in water for injection 100g, stirring and dissolving, for subsequent use after all dissolving;
(2) add 2.5g lysozyme raw material wherein, make its pH value to 6.3 ± 0.3;
(3) 4 DEG C of cold preservations leave standstill 2h, hang down and melt funnel filtration;
(4) add bentonite 2g, 80 DEG C of absorption 5min, sand stick is crossed and is filtered bentonite, obtains the lysozyme formulation after processing for subsequent use.
embodiment 3: the preparation of lysozyme formulation
(1) getting sodium sulfate 4.0g adds in water for injection 100g, stirring and dissolving, for subsequent use after all dissolving;
(2) 5.0g lysozyme raw material is added wherein, sodium hydroxide adjust ph to 8.0 ± 0.2;
(3) room temperature (25 DEG C) leaves standstill 6h, and sand stick filters;
(4) active carbon 10.0g is added, room temperature (25 DEG C) absorption 24h, after sand stick is crossed and is filtered active carbon, 10% soda lye adjust ph to 10.8 ± 0.3, isoelectric point, IP sedimentation lysozyme solid, solid collected by filtration, at 30 DEG C, namely vacuum drying 24h obtains lysozyme formulation.
embodiment 4: the preparation of lysozyme formulation
(1) getting 20.0g lysozyme raw material adds in water for injection 50g, stirring and dissolving, for subsequent use after all dissolving;
(2) add sodium citrate 0.2g wherein, make its pH value to 5.5 ± 0.2;
(3) 10 DEG C of standing 12h, in-depth filtration cardboard filter;
(4) add kieselguhr 2.0g, 10 DEG C of absorption 48h, sand stick obtains lysozyme formulation for subsequent use after crossing and filtering kieselguhr.
embodiment 5: the preparation of lysozyme formulation
(1) getting 10.0g lysozyme raw material adds in water for injection 100g, stirring and dissolving, for subsequent use after all dissolving;
(2) sodium chloride 5.0g is added wherein, triethanolamine adjust ph to 7.5 ± 0.3;
(3) 8 DEG C of standing 24h, filter element filtering;
(4) add active carbon 5.0g, 25 DEG C of absorption 12h, after sand stick is crossed and filtered active carbon, carry out spraying dry.
(5) spray-dired charging rate is 1.0ml/min, and inlet air flow temperature is generally 120 DEG C, and exit flow temperature is at 60 DEG C, and sprinkler pressure is at 1.2kg/cm
2, after drying, obtain lysozyme formulation.
Note: the sterling that the lysozyme raw material used in above-described embodiment 1-5 is lysozyme or be essentially sterling (can be considered sterling), wherein the content of lysozyme is about 10,000 U/mg.
embodiment 6-1: with the polyvinyl alcohol eye drop of lysozyme formulation as antibacterial
1) prescription:
2) technique:
(1) water for injection getting 70 DEG C-80 DEG C is appropriate, adds polyvinyl alcohol, stirs evenly, after making it fully swelling, for subsequent use.
(2) get appropriate water for injection, add boric acid, Borax, glycerol successively, it is rear for subsequent use that stirring makes it dissolve.
(3) lysozyme formulation that prepared by Example 2 adds in (2), for subsequent use after all dissolving.
(4) add in above-mentioned solution by swelling good polyvinyl alcohol, moisturizing, to full dose, stirs evenly and get final product.
embodiment 6-2: the polyvinyl alcohol eye drop using ethyl hydroxybenzoate as antibacterial
1) prescription:
2) technique:
(1) water for injection getting 70 DEG C-80 DEG C is appropriate, adds polyvinyl alcohol, stirs evenly, after making it fully swelling, for subsequent use.
(2) get appropriate water for injection, add boric acid, Borax, glycerol successively, it is rear for subsequent use that stirring makes it dissolve.
(3) getting ethyl hydroxybenzoate adds in (2), for subsequent use after all dissolving.
(4) add in above-mentioned solution by swelling good polyvinyl alcohol, moisturizing, to full dose, stirs evenly and get final product.
embodiment 7-1: the aspartic vitamin eye drops taking lysozyme formulation as antibacterial
1) prescription:
2) technique:
(1) get about 80g water for injection, after adding Naphazoline Chloridum, methyl-sulfuric acid neostigmine, chlorphenamine maleate, aspartic acid, glycyrrhizic acid dipotassium dissolving successively, add sodium chloride and dissolve.
(2) in above-mentioned solution, add lysozyme formulation prepared by embodiment 2, after dissolving, add water for injection to 100ml, to obtain final product.
embodiment 7-2: the aspartic vitamin eye drops being antibacterial with untreated lysozyme
1) prescription:
2) technique:
(1) get about 80g water for injection, after adding Naphazoline Chloridum, methyl-sulfuric acid neostigmine, chlorphenamine maleate, aspartic acid, glycyrrhizic acid dipotassium dissolving successively, add sodium chloride and dissolve.
(2), after adding untreated lysozyme powder dissolution in above-mentioned solution, add water for injection to 100ml, to obtain final product.
Except being applied to solution-type eye drop, lysozyme in eye suspensoid, Emulsion, also can be adopted as antiseptic and inhibiting bacteria function agent, to suppress the inner growth of microorganism of solution.Specifically be illustrated by following examples 3, embodiment 4.
embodiment 8-1: the compound neomycin sulphate suspension type using lysozyme formulation as antibacterial
eye drop
1) prescription:
2) technique:
(1) get suitable 70 DEG C of-80 DEG C of waters for injection, after adding hypromellose wherein, stir, make it completely swelling;
(2) separately get the lysozyme formulation that suitable water for injection adds sodium hydrogen phosphate, sodium dihydrogen phosphate, glycerol, embodiment 3 preparation wherein, after all dissolving, add polygynax, sulphuric acid polymyxin is for subsequent use after dissolving;
(3) (1) is added stirring and evenly mixing in (2), for subsequent use;
(4) prednisolone acetate is ground with after a small amount of above-mentioned solution-wet, after making it be uniformly dispersed, add in above-mentioned solution, stirring and evenly mixing;
(5) add water for injection to 100.0ml, stir evenly and get final product.
embodiment 8-2: the compound neomycin sulphate suspension type using phenoxyethanol as antibacterial drips
eye liquid
1) prescription:
2) technique:
(1) get suitable 70 DEG C of-80 DEG C of waters for injection, after adding hypromellose wherein, stir, make it completely swelling;
(2) separately get suitable water for injection and add sodium hydrogen phosphate, sodium dihydrogen phosphate, glycerol wherein, phenoxyethanol, after all dissolving, add polygynax, sulphuric acid polymyxin is for subsequent use after dissolving;
(3) (1) is added stirring and evenly mixing in (2), for subsequent use;
(4) prednisolone acetate is ground with after a small amount of above-mentioned solution-wet, after making it be uniformly dispersed, add in above-mentioned solution, stirring and evenly mixing;
(5) add water for injection to 100.0ml, stir evenly and get final product.
embodiment 9-1: the vitamin A ocular microemulsion taking lysozyme formulation as antibacterial
1) prescription:
2) technique:
(1) get suitable 50 DEG C of-60 DEG C of waters for injection, after adding hydroxyethyl-cellulose wherein, stir, make it completely swelling;
(2) get appropriate water for injection, add wherein disodium edetate, embodiment 5 prepare lysozyme formulation, glycerol, Tween 80 dissolve after, for subsequent use;
(3) the medium chain triglyceride acid esters of recipe quantity is got, after adding BHA dissolving wherein, after adding vitamin A mixing wherein, for subsequent use;
(4) (3) are under agitation slowly poured in (2), after mix homogeneously, add water for injection to 100ml, to obtain final product.
embodiment 9-2: the vitamin A ocular microemulsion taking benzalkonium chloride as antibacterial
1) prescription:
2) technique:
(1) get suitable 50 DEG C of-60 DEG C of waters for injection, after adding hydroxyethyl-cellulose wherein, stir, make it completely swelling;
(2) appropriate water for injection is got, after adding disodium edetate, benzalkonium chloride, glycerol, Tween 80 dissolving wherein, for subsequent use;
(3) the medium chain triglyceride acid esters of recipe quantity is got, after adding BHA dissolving wherein, after adding vitamin A mixing wherein, for subsequent use;
(4) (3) are under agitation slowly poured in (2), after mix homogeneously, add water for injection to 100ml, to obtain final product.
Lysozyme is except eye liquid preparation, and it also can be used as eye semi-solid preparation and uses as the antibacterial in the dosage form such as Eye ointments, gel for eye, is described in detail with experimental example 3,5.
embodiment 10-1: the ganciclovir ophthalmic gel taking lysozyme formulation as antibacterial
1) prescription:
2) technique:
(1) get suitable 70 DEG C of-80 DEG C of waters for injection, after adding hypromellose, carbomer wherein, stir, make it completely swelling;
(2) get appropriate water for injection, add successively wherein glycerol, ganciclovir dissolve after, the diluted hydrochloric acid dissolution embodiment 3 that separately takes a morsel preparation lysozyme formulation, add wherein stir evenly for subsequent use.
(3), after (2) are added stirring in (1), add appropriate triethylamine wherein, make its pH value within the scope of 6.0-7.0.After injecting water to 100ml, stir evenly and get final product.
embodiment 10-2: the ganciclovir ophthalmic gel taking thimerosal as antibacterial
1) prescription:
2) technique:
(1) get suitable 70 DEG C of-80 DEG C of waters for injection, after adding hypromellose, carbomer wherein, stir, make it completely swelling;
(2) appropriate water for injection is got, for subsequent use after adding glycerol, thimerosal, ganciclovir dissolving wherein successively.
(3), after (2) are added stirring in (1), add appropriate triethylamine wherein, make its pH value within the scope of 6.0-7.0.Inject with after water water to 100ml, stir evenly and get final product.
embodiment 11-1: the lidocaine hydrochloride eyes taking lysozyme formulation as antibacterial is solidifying
glue
1) prescription:
2) technique:
(1) after getting appropriate water for injection dissolving sodium alginate, for subsequent use.
(2) separately get suitable water for injection, add successively and dissolve sodium chloride, lysozyme formulation prepared by embodiment 5, after lidocaine hydrochloride, (1) added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
embodiment 11-2: the lidocaine hydrochloride eyes taking chlorobutanol as antibacterial is solidifying
glue
1) prescription:
2) technique:
(1) after getting appropriate water for injection dissolving sodium alginate, for subsequent use.
(2) separately get suitable water for injection, add successively and after dissolving sodium chloride, chlorobutanol, lidocaine hydrochloride, (1) added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
In addition, lysozyme can also be used as compound preservative jointly with other antibacterial, while reducing the consumption of other antibacterial, does not reduce antiseptic effect, thus is ensureing, under the condition that preparation is aseptic, can reduce again the injury to eye.Described in detail especially by experimental example 7.
embodiment 12-1: lysozyme formulation and benzalkonium chloride form the cattle sulphur that compound preservative is made
acid eye drop
1) prescription:
2) technique:
(1) after getting appropriate water for injection dissolving benzalkonium chloride, for subsequent use after all dissolving.
(2) separately get suitable water for injection, add lysozyme formulation that embodiment 1 obtains successively, after taurine dissolves, (1) added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
embodiment 12-2: using benzalkonium chloride separately as the taurine eye drops that antibacterial is made
1) prescription:
2) technique:
(1), after getting appropriate water for injection dissolving benzalkonium chloride, band is for subsequent use after all dissolving.
(2) separately get suitable water for injection, after adding Borax, taurine dissolving successively, (1) is added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
Visible above, in eye liquid preparation, semi-solid preparation, all can lysozyme as antibacterial, in addition, lysozyme can also in other ophthalmic preparations as antibacterial use, such as eye solid preparation etc., are not illustrated one by one at this.
embodiment 13: lysozyme formulation and ethyl hydroxybenzoate form the cromoglicic acid that compound preservative is made
sodium eye drop
1) prescription:
2) technique:
(1) after getting appropriate water for injection dissolving ethyl hydroxybenzoate, for subsequent use after all dissolving.
(2) separately get suitable water for injection, after adding the lysozyme formulation dissolving of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium cromoglicate, embodiment 1 preparation successively, (1) is added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
embodiment 14: the polyvidone eye drop that lysozyme formulation is made as antibacterial
1) prescription:
2) technique:
(1), after getting appropriate water for injection dissolving polyvidone, after all dissolving, 24h is soaked.
(2) separately get suitable water for injection, after adding the lysozyme formulation dissolving of sodium chloride, embodiment 3 preparation successively, (1) is added and wherein stirs.
(3) add water for injection to 100ml, to obtain final product.
embodiment 15: the stability experiment (1) of ophthalmic preparation and lysozyme
1. laboratory sample: the sample made at above-described embodiment 7-1,7-2.
2. experimental technique: the sample made by 7-1 and 7-2, fill is in plastics bottle for eyedrops.Long-time stability are investigated sampling in 0,3,6,9,12,18,24,36 month; Accelerate to investigate sampling in 0,1,2,3,6 month.Detect the character of ophthalmic preparation, visible foreign matters, pH value, osmotic pressure and lysozyme content.
3. experiment condition:
Long-time stability are investigated and sample are placed in temperature is 25 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 60% ± 10%.
Accelerate to investigate sample is placed in 40 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 20% ± 2%.
4. lysozyme content assay method:
Lysozyme content can react lysozyme activity, and unit is U/ml.Usual employing Determination method measures its content, and method is as follows:
This product 1ml is got in need testing solution preparation, puts in 100ml measuring bottle, adds phosphate buffer and (get sodium dihydrogen phosphate 10.4g, sodium hydrogen phosphate 7.86g, disodium edetate 0.37g, be dissolved in water and make to become 1000ml, adjust ph to 6.2) be diluted to scale, mix homogeneously.
The preparation of substrate suspension takes lyase micrococcus 10mg, add phosphate buffer 5, grind 3 minutes in mortar, add phosphate buffer more appropriate, cumulative volume is made to be about 100ml, suspension is in 25 ± 0.1 DEG C time, and the trap recorded at the wavelength place of 450nm is 0.7 ± 0.05 (prepared before use)
Assay method precision measures the substrate suspension 3ml of 25 ± 0.1 DEG C, puts in 1cm colorimetric pool, measures trap, as the reading A of zero second at the wavelength place of 450nm
0, then precision measures the need testing solution 0.15ml of 25 ± 0.1 DEG C, adds in above-mentioned colorimetric pool, mixes rapidly, with manual time-keeping, measures trap A again to when 60 seconds; Precision measures phosphate buffer 0.15ml simultaneously, with method operation, as blank assay, records the reading A of zero second
0' and the reading A ' of 60 seconds.
Computing formula:
Tire
5. experimental result: ophthalmic preparation and the lysozyme study on the stability of embodiment 7-1 and 7-2 contrast.
Table 1: the study on the stability contrast table of ophthalmic preparation and lysozyme
Can as apparent from above-mentioned experimental data, lysozyme before and after process is for character, pH value, the basic zero difference in osmotic pressure aspect of preparation, as can be seen from visible foreign matters, content aspect, the lysozyme stability after process is obviously better than the stability of untreated lysozyme.
embodiment 16: the stability experiment (2) of ophthalmic preparation and antibacterial
1. laboratory sample: at above-described embodiment 9-1,9-2 and the sample made.
2. experimental technique: the sample made by 9-1 and 9-2, fill is in plastics bottle for eyedrops.Long-time stability are investigated sampling in 0,3,6,9,12,18,24,36 month; Accelerate to investigate sampling in 0,1,2,3,6 month.Detect the character of ophthalmic preparation, visible foreign matters, pH value, osmotic pressure and antibacterial content.
3. experiment condition:
Long-time stability are investigated and sample are placed in temperature is 25 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 60% ± 10%.
Accelerate to investigate sample is placed in 40 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 20% ± 2%.
4. antibacterial content assaying method:
Lysozyme content can react lysozyme activity, and unit is U/ml.Usual employing Determination method measures its content, and method is as follows:
This product 1ml is got in need testing solution preparation, puts in 100ml measuring bottle, adds phosphate buffer and (get sodium dihydrogen phosphate 10.4g, sodium hydrogen phosphate 7.86g, disodium edetate 0.37g, be dissolved in water and make to become 1000ml, adjust ph to 6.2) be diluted to scale, mix homogeneously.
The preparation of substrate suspension takes lyase micrococcus 10mg, add phosphate buffer 5, grind 3 minutes in mortar, add phosphate buffer more appropriate, cumulative volume is made to be about 100ml, suspension is in 25 ± 0.1 DEG C time, and the trap recorded at the wavelength place of 450nm is 0.7 ± 0.05 (prepared before use)
Assay method precision measures the substrate suspension 3ml of 25 ± 0.1 DEG C, puts in 1cm colorimetric pool, measures trap, as the reading A of zero second at the wavelength place of 450nm
0, then precision measures the need testing solution 0.15ml of 25 ± 0.1 DEG C, adds in above-mentioned colorimetric pool, mixes rapidly, with manual time-keeping, measures trap A again to when 60 seconds; Precision measures phosphate buffer 0.15ml simultaneously, with method operation, as blank assay, records the reading A of zero second
0' and the reading A ' of 60 seconds.
Computing formula:
Tire
Benzalkonium chloride content assaying method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia 2010 editions annex VD)
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Be mobile phase with 0.005mol/L Spirit of Mindererus. (in every 1000ml containing triethylamine 10ml, with glacial acetic acid adjust ph to 5.0 ± 0.5)-acetonitrile (35: 65); Determined wavelength 214nm.
Algoscopy gets this product 20 μ l injection liquid chromatography, record chromatogram; It is appropriate that another precision takes benzalkonium chloride reference substance, is dissolved in water and quantitatively dilutes the solution made about containing 0.1mg in every 1ml.Be measured in the same method, test sample is as benzalkonium chloride, and by external standard method with calculated by peak area, benzalkonium chloride records result and represents with labelled amount.
5. experimental result: ophthalmic preparation and the lysozyme study on the stability of embodiment 9-1 and 9-2 contrast.
Table 2: the study on the stability contrast table of ophthalmic preparation and antibacterial
As apparent from above-mentioned experimental data can, the preparation in embodiment 9-1 and 9-2 is after acceleration investigation 6 months investigates 36 months with long-time stability, and preparation has no significant change in character, visible foreign matters, pH value, osmotic pressure, antibacterial content.All the quality of the pharmaceutical preparations can not be affected as antibacterial using benzalkonium chloride or lysozyme.
embodiment 17: the stability experiment (3) of ophthalmic preparation and antibacterial
1. laboratory sample: the sample made at above-described embodiment 10-1,10-2.
2. experimental technique: the sample made by 10-1 and 10-2, fill is in plastics bottle for eyedrops.Long-time stability are investigated sampling in 0,3,6,9,12,18,24,36 month; Accelerate to investigate sampling in 0,1,2,3,6 month.Detect the character of ophthalmic preparation, visible foreign matters, pH value, osmotic pressure and antibacterial content.
3. experiment condition:
Long-time stability are investigated and sample are placed in temperature is 25 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 60% ± 10%.
Accelerate to investigate sample is placed in 30 DEG C ± 2 DEG C, lucifuge is investigated under the condition of relative humidity 65% ± 5%.
4. antibacterial content assaying method:
Lysozyme content can react lysozyme activity, and unit is U/ml.Usual employing Determination method measures its content, and method is as follows:
This product 1ml is got in need testing solution preparation, puts in 100ml measuring bottle, adds phosphate buffer and (get sodium dihydrogen phosphate 10.4g, sodium hydrogen phosphate 7.86g, disodium edetate 0.37g, be dissolved in water and make to become 1000ml, adjust ph to 6.2) be diluted to scale, mix homogeneously.
The preparation of substrate suspension takes lyase micrococcus 10mg, add phosphate buffer 5, grind 3 minutes in mortar, add phosphate buffer more appropriate, cumulative volume is made to be about 100ml, suspension is in 25 ± 0.1 DEG C time, and the trap recorded at the wavelength place of 450nm is 0.7 ± 0.05 (prepared before use)
Assay method precision measures the substrate suspension 3ml of 25 ± 0.1 DEG C, puts in 1cm colorimetric pool, measures trap, as the reading A of zero second at the wavelength place of 450nm
0, then precision measures the need testing solution 0.15ml of 25 ± 0.1 DEG C, adds in above-mentioned colorimetric pool, mixes rapidly, with manual time-keeping, measures trap A again to when 60 seconds; Precision measures phosphate buffer 0.15ml simultaneously, with method operation, as blank assay, records the reading A of zero second
0' and the reading A ' of 60 seconds.
Computing formula:
Tire
Thiomersal assay method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia 2010 editions annex VD)
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; With 0.02mol/L Spirit of Mindererus .-methanol (70: 30) for mobile phase; Determined wavelength 240nm.
Algoscopy gets this product 20 μ l injection liquid chromatography, record chromatogram; It is appropriate that another precision takes thimerosal reference substance, is dissolved in water and quantitatively dilutes the solution made about containing 40 μ g in every 1ml.Be measured in the same method, test sample is as thimerosal, and by external standard method with calculated by peak area, thimerosal records result and represents with labelled amount.
5. experimental result: ophthalmic preparation and the lysozyme study on the stability of embodiment 10-1 and 10-2 contrast.
Table 3: the study on the stability contrast table of ophthalmic preparation and antibacterial
As apparent from above-mentioned experimental data can, the preparation in embodiment 10-1 and 10-2 is after acceleration investigation 6 months investigates 36 months with long-time stability, and preparation has no significant change at character, visible foreign matters, pH value, osmotic pressure.In antibacterial content, lysozyme content is substantially unchanged in investigation process, and thimerosal presents downward trend in investigation process.
embodiment 18: inhibitory effect is tested
For checking lysozyme is as the inhibitory effect of eye antibacterial, inventor measures with reference to the inhibitory effect of Chinese Pharmacopoeia 2010 editions X IX N antibacterial efficacy test method guidelines to above-described embodiment.
1. Experimental agents: the ophthalmic preparation prepared in each embodiment above.
2. experimental technique:
2.1 tested strains:
Pseudomonas aeruginosa (pseudomonas aeruginosa) (CMCC (B) 10 104)
Escherichia coli (Escherichia coli) (CMCC (B) 44 102)
Staphylococcus aureus (Staphylococcus aureus) (CMCC (B) 26 003)
Candida albicans (Candida albicans) (CMCC (F) 98 001)
Aspergillus niger (Aspergillus niger) (CMCC (F) 98 003)
2.2 test methods:
Get test sample 5 parts, be transferred to respectively in 5 suitable sterile chambers, a kind of test organisms inoculated by often kind of container, and quantity of microorganism inoculated is 10
5-10
6cfu, the volume of inoculation bacterium liquid must not exceed the 0.5%-1% of test sample volume, and fully mix, the test organisms in construction food is uniformly distributed.Then the test sample in explanation puts 20 DEG C-25 DEG C at experimental session, stored protected from light.
2.3 survival Counting alive microbial
Just inoculate (when 0) and the 7th day, 14 days and 28 natural gift at test sample and get test sample indescribably, measure contained bacterium number in every part of test sample with Plating or membrane-filter procedure.According to all belonging to measurement result, calculating bacterium number and the bacterium number of each interval time added by each test organisms of 1ml (g) test sample, and being converted into 1g value.
2.4 experimental result criterions
With reference to Chinese Pharmacopoeia 2010 editions, ophthalmic preparation belongs to 1 class test sample.
Decline to antiseptic effect prescribed bacteria 7 days bacterium numbers of wherein antibacterial and be no less than 1.0lg, 14 days bacterium numbers decline and are no less than 3.0lg, and within 14 days to 28 days, bacterium number does not increase; Fungus and initial value ratio, within 7,14,28 days, bacterium number does not all increase.By this regulation, lysozyme can meet the requirement of ophthalmic preparation to antibacterial.
3. experimental result.Respectively as shown in table 2-8 below.
3.1 make sample with embodiment 6-1 and embodiment 6-2, compare the antiseptic power of lysozyme and ethyl hydroxybenzoate, and result is as following table 4:
Table 4: clump count and Comparative result table in the antiseptic effect of lysozyme and ethyl hydroxybenzoate
As can be seen from the above table, the antiseptic effect no significant difference of lysozyme and ethyl hydroxybenzoate.
3.2 make sample with embodiment 7-1 and embodiment 7-2, compare the antiseptic power before and after lysozyme process, and result is as following table 5:
Table 5: clump count and Comparative result table in lysozyme process and untreated antiseptic effect
Untreated lysozyme and processed lysozyme are to the basic zero difference of inhibitory effect of escherichia coli and staphylococcus aureus as can be seen from the above table, but to the false single bleb bacterium of Aerugo and fungus, untreated lysozyme inhibitory effect is a little less than the lysozyme after process.
In addition, as can be seen from upper table result, lysozyme concentration is (minimum effective drug concentration) 0.3% time, and its inhibitory effect can meet national standard.
3.3 compared for the antiseptic power of lysozyme and phenoxyethanol in suspension type eye drop with embodiment 8-1 and embodiment 8-2, and result is as following table 6:
Table 6: the antiseptic effect comparing result table of lysozyme and phenoxyethanol
The inhibitory effect no significant difference of lysozyme and phenoxyethanol as can be seen from the above table.
3.4 compared for the antiseptic power of lysozyme and benzalkonium chloride in nourishing type eye drop with embodiment 9-1 and embodiment 9-2, and result is as following table 7:
Table 7: the antiseptic effect comparing result table of lysozyme and benzalkonium chloride
The antiseptic effect of benzalkonium chloride in its opthalmological usual amounts obviously will lower than the effect of lysozyme as can be seen from the above table.And due to lysozyme be human body endogenous material, under larger use amount on human body also without impact.Make nourishing type eye drop while eliminating is to the injury of human body eye table, the gnotobasis of its eye drop inside can be ensured.
3.5 compared for the antiseptic power in Eye ointments at middle lysozyme and thimerosal with embodiment 10-1 and embodiment 10-2, and result is as following table 8:
Table 8: the antiseptic effect comparing result table of lysozyme and thimerosal
Lysozyme and thimerosal inhibitory effect no significant difference as can be seen from the above table.
3.6 with embodiment 11-1 and embodiment 11-2 compared for middle lysozyme and chlorobutanol in instant gel for eye antiseptic power, result is as following table 9:
Table 9: the antiseptic effect comparing result table of lysozyme and chlorobutanol
The inhibitory effect of lysozyme and chlorobutanol there is no significant difference as can be seen from the above table.
3.7 antiseptic powers when compared for embodiment 12-1 and embodiment 12-2 the benzalkonium chloride and lysozyme and low concentration benzalkonium chloride compound use that are used alone high concentration contrast, and result is as following table 10:
Table 10:12-1 and 12-2 inhibitory effect comparing result table
As seen from the above table, when lysozyme and benzalkonium chloride compound use, can when benzalkonium chloride consumption reduce, while reducing the side effect of benzalkonium chloride, there is not significant change in inhibitory effect.
To sum up experimental result is visible, and compared with the conventional antibacterial such as benzalkonium chloride, the inhibitory effect of lysozyme and other antibacterial is without significant difference.And can share with other antibacterial, reduce its consumption, thus reduce the toxic and side effects of other antibacterial.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (18)
1. a preparation method for refining lysozyme, comprises the steps:
1) lysozyme is dissolved in water for injection, obtains lysozyme soln, in described lysozyme soln, the concentration of lysozyme is 0.3%-10% (w/w);
2) to step 1) in lysozyme soln in add stabilizing agent salt, stabilizing agent salt content is 5%-30% (w/w);
3) pH is regulated to be 5.5-6.6;
4) leave standstill, make lysozyme fully contact rear filtration with stabilizing agent salt, obtain filtrate; Described rest on 0 DEG C of-40 DEG C of environment under carry out, time of repose is between 2-48 hour;
5) add electronegative adsorbent to adsorb the impurity in filtrate, obtain the solution of clear, then cross and filter adsorbability material; Described adsorbent be selected from active carbon, kieselguhr and bentonite one or more; The use amount of described adsorbent is 0.001%-10% (w/w), and adsorption time is 5min-48h.
2. method according to claim 1, wherein, in described lysozyme soln, the concentration of lysozyme is 0.5%-5% (w/w).
3. method according to claim 1, wherein, described stabilizing agent salt be selected from sodium carbonate, sodium bicarbonate, sodium citrate, boric acid, Borax, potassium dihydrogen phosphate, potassium chloride and calcium chloride one or more.
4. method according to claim 1, wherein, described stabilizing agent salt be selected from boric acid, Borax, sodium citrate and dipotassium hydrogen phosphate one or more.
5. method according to claim 1, wherein, described pH is 5.5-6.5.
6. method according to any one of claim 1 to 5, it also comprises the steps:
Refining lysozyme is made refining lysozyme pressed powder.
7. method according to claim 6, wherein, uses lyophilization or the obtained refining lysozyme pressed powder of spraying dry.
8. claim requires the refining lysozyme that the method according to any one of 1 to 7 is obtained.
9. refining lysozyme according to claim 8 is preparing the purposes in ophthalmic preparation.
10. purposes according to claim 9, wherein, described ophthalmic preparation is eye drop or eye solid preparation.
11. purposes according to claim 9, wherein, described ophthalmic preparation is Eye ointments or gel for eye.
12. purposes according to claim 9 or 10 or 11, wherein, the content of the lysozyme in described ophthalmic preparation is 0.001%-25% (w/w).
13. purposes according to claim 9 or 10 or 11, wherein, the content of the lysozyme in described ophthalmic preparation is 0.01%-20% (w/w).
14. purposes according to claim 9 or 10 or 11, wherein, the content of the lysozyme in described ophthalmic preparation is 0.1%-15% (w/w).
15. purposes according to claim 9 or 10 or 11, wherein, the content of the lysozyme in described ophthalmic preparation is 0.3%-10% (w/w).
16. purposes according to claim 9 or 10 or 11, wherein, the content of the lysozyme in described ophthalmic preparation is 0.5%-5% (w/w).
17. refining lysozyme according to claim 8 are preparing the purposes in antibacterial and/or antiseptic.
The purposes of 18. refining lysozyme according to claim 8 in the antibacterial preparing ophthalmic preparation and/or antiseptic.
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