CN107744529A - A kind of eye-drops preparations of the extraction active factors containing probiotics and preparation method thereof - Google Patents
A kind of eye-drops preparations of the extraction active factors containing probiotics and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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Abstract
The present invention provides a kind of eye-drops preparations of the extraction active factors containing probiotics and preparation method thereof.The eye-drops preparations includes the EM factors 1% 20%, VB family vitamins 0.05 0.3%, taurine 0.5 3%, sodium chloride 0.5 1% and the 100ppm of menthol 20, and is mended by water for injection to 100%.The eye-drops preparations of the extraction active factors containing probiotics of the present invention, has the advantages that：Preservative is not added, avoids injury of the preservative to eye；The active component EM factors have the function that well anti-oxidant and resist damage of the light to cell；Being capable of the nutrient such as replenishing vitamins.
Description
Technical field
The invention belongs to biomedicine field, and in particular to it is a kind of containing probiotics extraction active factors eye-drops preparations and its
Preparation method.
Background technology
China is the higher country of Shortsightedness prevalence, from knowledge type crowd from, its incidence is more than 60%.It is another
Aspect is influenceed by modern office feature, and the development in aging society, and the incidence of xerophthalmia is with annual 10% speed
Degree is incremented by, so as to the main consumer group for constituting ophthalmic anti-inflammatory, moistening mesh medication.
At the same time, cataract of old people, glaucoma have the trend gradually risen, so as to objectively create huge eye
Section's medication market, the growth of cost index is paid along with health care, medical expenses, and ophthalmic remedy level is also improving constantly.
Under the advertising education of famous brand name, the eye health consciousness enhancing of consumer, got over for sub-health state
More to pay close attention to, the use to Related product is also more and more common, so that sale of the direct pull similar drug on retail market；
Meanwhile the price of ophthalmic product constantly rises, the expansion of the market in general marketing scale is promoted to a certain extent.Above-mentioned two
Individual factor is to directly result in the driving force of ophthalmic remedy retail terminal market rapid growth.
Medicament for the eyes market is the market of a sustainable development, is confirmed from authoritative data, at present the illness in eye market in general rule in China
Mould is 10,000,000,000 RMB；With the working environment of people and the change of living environment, rapid growth is all presented in numerous illness in eye
The impetus, online, the popularization of computer office cause the demand for alleviating visual fatigue using eye drops increasing, and with annual 25%
Ratio increase.The patient populations such as cataract are on the rise phenomenon and numerous tech electronic products make with astogeny
With, cause patient populations every year with million number increasing, this phenomenon is suddenly to be developed by the great potential for expediting the emergence of medicament for the eyes market again.
The ophthalmic remedy consumer of retail pharmacies is mainly to mitigate based on the symptoms such as visual fatigue, xerophthalmia, mild inflammation.Pin
The product sold mainly with improving eyesight, fatigue-relieving, nutrition is moistened, cleaning is escorted, antibacterial bacteriostatic, the only puckery eye health product such as antipruritic
Based on, traditional Chinese medicine eye drops account for larger proportion wherein.
Current domestic eyedrops is primarily present problems with：
(1) preservative will trigger a series of eye diseases to the long-time stimulus of eyes
Because easy bacterial infection after eyedrops is opened, can in eye drops and eyedrops in national relevant regulations
So that mark will not be made containing a small amount of preservative, but generally for preservative,.After consumer uses eyedrops, although can be temporarily
It is dry and astringent to improve eyes, but long-term use can influence human body itself lacrimal secretion, and the preservative in eyedrops can also change eyeball table
The environment in face, it is unfavorable to cornea, conjunctiva health commonly using meeting, trigger conjunctiva and corneal injury, eyes is produced dependence, draw
Send out eye disease.In addition to inflammation of eye section caused by microorganism, it is due to preservative on eye also to have significant component of inflammation
Caused by the long-time stimulus of chrotoplast.
(2) oxidative damage of retina is the principal element for causing glaucoma
The research clinically on glaucoma pathogenesis is focused primarily upon in the local anatomical structure of eyeball at present, and
In preclinical medicine, research shows that oxidative stress is one of important mechanisms for causing glaucoma to be fallen ill；Meanwhile there are some researches show
The generation of glaucoma with retina active oxygen metabolism is unbalance has certain relation.
(3) VDT syndromes
VDT syndromes are the syndrome that optic terminal display is brought, and are also office's eye syndrome.VDT is video
Display terminal, it is English " visual display terminal " abbreviation, including the display of television set, computer, electronics
Screen, game machine etc., it is seen everywhere in our various workplaces and social life.Influences of the VDT to operator's health
It has been be recognized that, operator may occur in which eye and general malaise, including eye, hand, shoulder, foot, lumbar fatigue, referred to as VDT symptom groupings.It is beautiful
The people of the studies have shown that long-term use computer of state's University of California's optical system generally suffers from VDT xerophthalmia, i.e., easy eye is dry, furious
With it is tired, present 10,000,000 people in the U.S. is affected by it.Estimate that this number of China is not less than 20,000,000.Regarding for VDT worker is tired
Labor is 4 times of common worker, on infringements of the VDT to eyes, what Lv Jie write《The most easy visual fatigue of computer people》One text has very detailed
Thin research.As information technology is widely available, the chance that people contact above-mentioned terminal display screen is increasing, is handling official business daily
Room work is also increased year by year using video display terminal (vdt) number.The easy excess eye-using of these people, especially for a long time, closely note
Depending on flicker or dull, dazzling screen etc., it can cause visual fatigue.
Therefore a good eye-drops preparations generally requires following functions：
(1) injury of preservative free or mitigation preservative
(2) it is anti-oxidant
(3) damage of the light to cell is mitigated
(4) nutrient such as replenishing vitamins
(5) give people to bring refrigerant sensation.
Solution method has following several at present：
(1) product of preservative free is sterile preparation, i.e. single bottle is intended for single use, and this causes price very expensive, commonly disappears
Expense person can not receive, therefore more than 90% eye-drops preparations still contains preservative in the market.Such as auspicious droplet ocular fluid, using poly-
Vinyl alcohol simulates tear, can alleviate the generation of xerophthalmia, but it is even have an irritated eyes and allergic reaction, allergic constitution person is careful
With.
(2) by adding the antioxidant contents such as VE, resveratrol.But resveratrol is oil-soluble, not soluble in water, thus one
As can not be used in ophthalmic preparation, there is German company to be added to by technologies such as liposomes in aqua at present, but
Due to its less stable, the shelf term of validity is usually no more than 1 year.
(3) VDT syndromes are directed to, typically barrier is provided using artificial tears, mitigates the generation of xerophthalmia, while eye of releiving
Portion's fatigue.But killing of the shortwave such as the ultraviolet and blue light light for ocular cell is also very important.Cataract belongs to
Multi-factor disease, but zoopery and epidemiology survey be all it has been shown that ultraviolet belongs to one of high risk factor of cataract,
And it is also possible to increase the risk of macular degeneration.
The content of the invention
In view of the above the shortcomings that prior art, the first object of the present invention is to provide a kind of sterile no corrosion-resistant eye
Use preparation.
To achieve the above object, the invention provides a kind of eye-drops preparations of the extraction active factors containing probiotics, including such as
Lower component：
Wherein, the EM factors are the extract solution of probiotics, can specifically be obtained by the following method：
(1) probiotics is placed in solvent and obtains mixed liquor, probiotics is cracked using physical method, and centrifuged, take supernatant；
(2) supernatant addition anion-exchange column is eluted, collects each component respectively；
(3) detect and remove the component of no polysaccharide and the component containing protein, i.e. crude product；
(4) crude product is further purified.
Preferably, the eye-drops preparations of the extraction active factors containing probiotics, including following component：
One of innovative point of the present invention is embodied in be found when the EM factors are applied to eye with good effect first.
Further, the eye-drops preparations of the extraction active factors containing probiotics, including following component：
Above-mentioned percentage is mass percent.
Preferably, the VB family vitamins are VB6And VB12, wherein VB6And VB12Mass ratio be 5:1~1:1.
Preferably, the probiotics is selected from Bifidobacterium (Bifidobacterium), Lactobacillus
(Lactobacillus) any one or a few or in Bacteroides (Bacteroides)；More preferably, the probiotics choosing
From Bifidobacterium (Bifidobacterium).
The step (1) also contains any one or a few in following characteristics：
Preferably, the percetage by weight of bacterium is 5%~45% in the mixed liquor；Further, bacterium in the mixed liquor
Percetage by weight be 8%~45%；
Preferably, the method for the cracking is selected from the cracking of ultrasonic treatment instrument, historrhexis's instrument cracks, high pressure homogenizer splits
Solution or chemical method cracking in any one or a few；
Preferably, the condition of the centrifugation is 8000g~30000g/ points, and the time is 10~60 minutes；
Preferably, will centrifuge obtain sediment again using physical method crack, take supernatant after centrifugation, will twice from
Supernatant after the heart merges.
The solvent can be selected from any one in water, sodium phosphate aqueous solution.
Preferably, the step (2) also contains any one or a few in following characteristics：
Preferably, 10-300mM pH are selected from using equilibrium liquid adjustment anion-exchange column, the equilibrium liquid before the elution
6.5-8.5 Tris-HCl (three (methylol) aminomethanes) solution or 10-200mM pH 6-9 phosphoric acid solution；
Preferably, the eluent is molten from 10-300mM Tris-HCl/1M sodium chloride solutions and 10-200mM phosphoric acid
Liquid/1M sodium chloride solutions；PH is 6.0-8.5；
Preferably, specific rotation detector, refractive index detection device or ultraviolet detection are selected using detector during the elution
Any one in device.
Detected in the step (3) and remove the component of no polysaccharide and the component containing protein, can used existing
Technology, total sugar content is surveyed for example with phenolsulfuric acid method, and total protein is surveyed using BCA protein reagents box.
For further optimizing design scheme, step (3) also includes the protein removal in crude product, it is furthermore preferred that adopting
Protein is removed with molecular exclusion chromatography method.
Preferably, the step (4) further purifies crude product including crude product is passed through into molecular exclusion chromatography and filter membrane institute
State Tris-HCl solution or 10-200mM pH 6-9 that equilibrium liquid in molecular exclusion chromatography is selected from 10-300mM pH 6.5-8.5
Phosphoric acid solution；Collect polysaccharide component and be less than or equal to 0.22 μm of filter membrane by aperture.
The second goal of the invention of the present invention is to provide a kind of preparation side of the eye-drops preparations of the extraction active factors containing probiotics
Method.
For achieving the above object, the invention provides a kind of system of the eye-drops preparations of the extraction active factors containing probiotics
Preparation Method, comprise the following steps：After each raw material components are well mixed, purify, removed in possible by 767 active carbon adsorptions
Toxin；Centrifugation removes activated carbon, is filtered off after 0.22 micron membranes except residual impurity and bacterium；And then dispensed, Ran Hougao
Warm and humid heat sterilization, is finally packed.
Preferably, the preparation method is carried out in ten thousand grades of cleanliness factor environment.
As described above, the eye-drops preparations of the extraction active factors containing probiotics of the present invention, has the advantages that：Do not add
Adding preservative agent, so as to avoid injury of the preservative to eye；The active component EM factors have well anti-oxidant and resist light
The effect of damage of the line to cell；Being capable of the nutrient such as replenishing vitamins.
Brief description of the drawings
Fig. 1 is people's epidermal keratinocyte figure after rhodamine dyeing, and its fluorescence intensity and mitochondrial transmembrane potentials are into positive
Close；Wherein Control is control；H2O2For dioxygen water process；EM is 1% concentration EM factor treatments；EM+H2O2For the EM factors and
Hydrogen peroxide coprocessing.
Fig. 2 behaviour epidermal keratinocyte mitochondrial transmembrane potentials percentage block diagrams.
The adjustment effect figure to P-53 gene expression amounts in human lens epithelial cells of Fig. 3 different amounts EM factors.
Embodiment
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.It should be clear that the process equipment or device that are not indicated specifically in the following example
Use conventional equipment or device in the art.In addition, it is to be understood that one or more method and steps mentioned in the present invention are simultaneously
Other method step can also be present or may be used also between the step of these are specifically mentioned by not repelling before and after the combination step
To insert other method step, unless otherwise indicated；It should also be understood that one or more equipment/devices mentioned in the present invention it
Between combination annexation do not repel and can also have other equipment/device before and after the unit equipment/device or at this
Other equipment/device can also be inserted between the two equipment/devices specifically mentioned a bit, unless otherwise indicated.It is moreover, unless another
It is described, the numbering of various method steps is only to differentiate the convenient tool of various method steps, rather than the row for limitation various method steps
Row order limits the enforceable scope of the present invention, and its relativeness is altered or modified, without essence change technology contents
In the case of, when being also considered as the enforceable category of the present invention.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
Explicitly point out in addition, singulative "one", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring
Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic
updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe,
CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；
METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.),
Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119,
Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1：
Bifidobacterium breve (Bifidobacterium breve) dry bacterium powder is mixed into obtain mixed liquor, mixed liquor with sterilized water
The weight of middle bacterium is 8% (w/w).Above-mentioned mixed liquor is cracked with ultrasonic treatment instrument.By the bacterium solution that this was cracked with from
Scheming was with the centrifugation of 10,000g, 30 minutes and collected supernatant, by insoluble bacteria lysis fragment and uncracked bacterium again
It is secondary to be cracked using ultrasonic treatment instrument, it is then centrifuged for, collects supernatant.The supernatant for collecting acquisition twice is merged.
The supernatant of above-mentioned collection is diluted with isometric 20mMpH8.0Tris-HCl solution.By the supernatant of the dilution
Liquid passes through anion-exchange column.Before loading, anion exchange is lived to be adjusted with 10mM pH 8.0 Tris-HCl equilibrium liquids.
After equilibrium liquid adjusts exchange column, sample is added, the component not combined with equilibrium liquid elution with exchange column, Ran Houyong
The eluent of 10mMTris-HCl/1M sodium chloride solutions carries out linear gradient elution, collects each component respectively (using ultraviolet
Detector, peak is shown as by UV260 and UV280).
Each component collected, total sugar content is surveyed with phenolsulfuric acid method, and total protein is surveyed with BCA protein reagents box.Go
Except without polysaccharide or having the component of albumen, by Fraction collection of the others containing only polysaccharide and polysaccharide component is incorporated as.
By the polysaccharide component of the collection by molecular exclusion chromatography, the Tris-HCl that equilibrium liquid is selected from 10mM pH 8.5 is molten
Liquid, and insoluble impurities is removed by 0.22 μm of membrane filtration.
Embodiment 2
By bifidobacterium longum (Bifidobacterium longum) dry bacterium powder and 20mM pH 8.5 sodium phosphate aqueous solution
Mixed liquor is mixed to obtain, and makes the weight 15% (w/w) of bacterium in mixed liquor.Above-mentioned bacterium solution is cracked with historrhexis's instrument.Will
The bacterium solution centrifuge cracked collects supernatant respectively with 13,000g centrifugation 25 minutes.Remove insoluble bacterium
Cleaved fragment and uncracked bacterium.
Supernatant is passed through into anion-exchange column.Before loading, the sodium phosphate with 20mM pH8.5 is lived in anion exchange
Solution is adjusted, and adds sample, linear gradient elution is carried out with 20mM sodium phosphates/1M sodium chloride solutions pH 8.5 eluent, point
Each component (using UV-detector, peak is shown as by UV260 and UV280) is not collected.
Each component collected surveys total reducing sugar with phenolsulfuric acid method and surveys total protein with BCA protein reagents box.Remove nothing
Polysaccharide or the peak/component for having albumen, other peaks are collected and are incorporated as polysaccharide component.
The polysaccharide component of the collection is selected from 100mM pH by molecular exclusion chromatography desalination, equilibrium liquid and eluent
6.5Tris-HCl solution simultaneously removes insoluble impurities by 0.22 μm of membrane filtration.
Embodiment 3
Bifidobacterium bifidum (Bifidobacterium bifidum) dry bacterium powder is mixed into obtain mixed liquor with sterilized water, and
Make the weight 12% (w/w) of bacterium in mixed liquor.Above-mentioned bacterium solution is cracked with high pressure homogenizer.The bacterium solution that this was cracked is used
Centrifuge collects supernatant with 20,000g centrifugation 10 minutes, and place to go is except insoluble bacteria lysis fragment and uncracked
Bacterium.
The supernatant of above-mentioned collection is diluted with isometric 20mM pH8.2 sodium radio-phosphate,P-32 solutions.By the supernatant of the dilution
Pass through an anion-exchange column.Before loading, the sodium radio-phosphate,P-32 solution with 10mM pH8.2 is lived in anion exchange, when sample is attached
After, linear gradient elution is carried out with 10mM sodium phosphates/1M sodium chloride solutions pH 8.2 eluent, collects each respectively
Component or peak (using UV-detector, peak is shown as by UV260 and UV280).
Each component collected, survey total reducing sugar with phenolsulfuric acid method and survey total protein with BCA protein reagents box.Remove
Sugar-free or the peak/component for having albumen, other peaks are collected and are incorporated as polysaccharide component.
The polysaccharide component is added in the 5M sodium chloride solutions that volume is polysaccharide component 1/4, makes last sodium chloride dense
Degree is more than or equal to 1M.And the component containing polysaccharide is further separated by hydrophobic chromatography.Equilibrium liquid is with 10mM pH
8.2 sodium phosphates/1M sodium chloride solutions, and collect the component of non-cohesive hydrophobic chromatography post.After the attachment of sample other components, use
10mM pH 8.2 sodium radio-phosphate,P-32 solution carries out linear gradient elution, according to conductance, collect prior to 600mM sodium chloride (according to point
Determined from the conductive linearity curve shown in equipment) polysaccharide component of elution, and merged with unattached component as effective
Total saccharic composition.
Above polysaccharide component is selected from 50mM pH 8 Tris-HCl solution by molecular exclusion chromatography desalination, equilibrium liquid,
And by the way that 0.22 μm of membrane filtration is degerming and other insoluble impurities.
Embodiment 4
A kind of eye-drops preparations of the extraction active factors containing probiotics, including：
Embodiment 5
A kind of eye-drops preparations of the extraction active factors containing probiotics, including：
Embodiment 6
A kind of eye-drops preparations of the extraction active factors containing probiotics, including：
Embodiment 7
Come really by determining oxygen radical absorbability (oxygen radical absorbance capacity, ORAC)
Determine the oxidation resistance of the EM factors.By measuring and calculating, 3.5 μ g/ml Trolox water soluble vitamin of the EM factors equivalent to 150 μM
E oxidation resistance (table 1), illustrate that the EM factors have stronger antioxidant activity in vitro.The EM factors used in table 1 are according to reality
The method of example 1 is applied to be prepared.
The oxidation resistance of the table 1.EM factors assesses (ORAC)
Embodiment 8
The ability that EM factor pair epidermal keratinocytes resist hydrogen peroxide induced injury is inventors tested a larged, such as Fig. 1 and 2 institutes
Show, nerve cell is individually handled 4 hours with 31.25 μ g/ml hydrogen peroxide, obvious mitochondrial transmembrane potentials drop can be induced
Low, and individually add the EM factors of 1% concentration, without the apoptosis for causing cell, (control group and EM treatment groups do not have significance difference
It is different, p>0.05), therefore, the EM factors are in itself safe.And by the EM factors and H2O2(EM+H when being added in cell2O2
Group), the EM factors can significantly improve the oxidation resistance (H of cell2O2Treatment group) (p<0.05).Therefore, the EM factors also have
The function for the cell oxidative damage that effect protection hydrogen peroxide induces.The EM factors used of embodiment 8 are according to the method system of embodiment 2
It is standby to obtain.
Embodiment 9
Inventor has inquired into EM factor pairs p53 transcription water using UVB irradiation human lens epithelial cells systems (SRA01/04)
Flat regulation and control, and then confirm the inhibitory action of EM factor pair Apoptosis.As shown in figure 3, pre-processed 8 hours by the EM factors
Human lens epithelial cells system (SRA01/04) UVB irradiate 10 minutes after, p53 mrna expression amount is substantially less than untreated
The cell of group.Therefore, the EM factors have the function of effectively suppressing Apoptosis.The EM factors used of embodiment 9 are according to implementation
The method of example 3 is prepared.
Embodiment 10
The collyrium 5ml being prepared by the method for embodiment 6 is taken, pours into standby silica gel cup, then by silica gel cup back-off
To objective eye, 15min is cleaned, cup is then removed, the intraocular impurity washed out is can see in cup.Eye is contacted by the EM factors
Ball surrounding enviroment, peroxide can be removed, while the generation to reduce inflammation, reduce red blood silk.
Embodiment above is to illustrate embodiment disclosed by the invention, can not be interpreted as the limit to the present invention
System.In addition, in various modifications and invention listed herein method, composition change, do not departing from the scope of the present invention
Be obvious for those skilled in the art on the premise of spirit.Although a variety of specific of the present invention has been combined
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various, obvious modification should all include to obtain invention for those skilled in the art as described above
Within the scope of the invention.
Claims (10)
1. a kind of eye-drops preparations of the extraction active factors containing probiotics, it is characterised in that including following component：
Wherein, the EM factors are the extract solution of probiotics.
2. the eye-drops preparations of the extraction active factors containing probiotics as claimed in claim 1, it is characterised in that including such as the following group
Point：
3. the eye-drops preparations of the extraction active factors containing probiotics as claimed in claim 1, it is characterised in that including such as the following group
Point：
4. the eye-drops preparations of the extraction active factors containing probiotics as described in any one of claims 1 to 3, it is characterised in that institute
The EM factors are stated to obtain by the following method：
(1) probiotics is placed in solvent and obtains mixed liquor, probiotics is cracked using physical method, and centrifuged, take supernatant；
(2) supernatant addition anion-exchange column is separated, collects each component respectively；
(3) detect and remove the component of no polysaccharide and the component containing protein, i.e. crude product；
(4) crude product is further purified by hydrophobic chromatography, molecular exclusion.
5. the eye-drops preparations of the extraction active factors containing probiotics as claimed in claim 4, it is characterised in that the probiotics choosing
From any one or a few in Bifidobacterium, Lactobacillus or Bacteroides.
6. the eye-drops preparations of the extraction active factors according to claim 4 containing probiotics, it is characterised in that：The step
(1) percetage by weight of bacterium is 5%~45% in mixed liquor in；
The method of the cracking is selected from the physical methods such as the cracking of ultrasonic treatment instrument, historrhexis's instrument cracking or high pressure homogenizer and split
Any one or a few in solution mode；
The condition of the centrifugation is 8000g~30000g/ points, and the time is 10~60 minutes.
7. the eye-drops preparations of the extraction active factors containing probiotics as claimed in claim 4, it is characterised in that the step (2)
Before middle elution 10-300mM, pH 6.5-8.5 Tris- are selected from using equilibrium liquid adjustment anion-exchange column, the equilibrium liquid
The phosphoric acid solution of HCl solution or 10-200mM pH 6-9；
The eluent is from 10-300mM Tris-HCl/1M sodium chloride solutions or 10-200mM phosphoric acid solution/1M sodium chloride
Solution；PH is 6.0-8.5.
8. the eye-drops preparations of the prebiotic extraction active factors containing probiotics as claimed in claim 4, it is characterised in that the step
(4) by crude product further by being further purified including molecular exclusion chromatography and filter membrane, equilibrium liquid in the molecular exclusion chromatography
The phosphoric acid solution of Tris-HCl solution or 10-200mM pH 6-9 selected from 10-300mM pH 6.5-8.5；Collect polysaccharide component
And it is less than or equal to 0.22 μm of filter membrane by aperture.
9. a kind of preparation method of the eye-drops preparations of extraction active factors containing probiotics as described in any one of claims 1 to 3,
Comprise the following steps：After each raw material components are well mixed, are purified by active carbon adsorption, remove possible endotoxin；Centrifugation
Activated carbon is removed, is filtered off after 0.22 micron membranes except residual impurity；And then dispensed, then high-temperature heat sterilization, finally
Packed.
10. the preparation method of the eye-drops preparations of the extraction active factors containing probiotics as claimed in claim 9, it is characterised in that
The preparation method is carried out in ten thousand grades of cleanliness factor environment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112057478A (en) * | 2020-10-16 | 2020-12-11 | 常州市艾斯康生物医药有限公司 | Pharmaceutical preparation for relieving and preventing allergic rhinitis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106977617A (en) * | 2017-04-12 | 2017-07-25 | 常州谙美生物科技有限公司 | There is uvioresistant in probiotics, sensitive skin and the polysaccharide and its separation method of anti-senescence function and application is repaired |
| CN107028973A (en) * | 2017-05-12 | 2017-08-11 | 浙江工贸职业技术学院 | Eye drops for relieving asthenopia and preparation method thereof |
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2017
- 2017-10-25 CN CN201711005777.7A patent/CN107744529A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106977617A (en) * | 2017-04-12 | 2017-07-25 | 常州谙美生物科技有限公司 | There is uvioresistant in probiotics, sensitive skin and the polysaccharide and its separation method of anti-senescence function and application is repaired |
| CN107028973A (en) * | 2017-05-12 | 2017-08-11 | 浙江工贸职业技术学院 | Eye drops for relieving asthenopia and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112057478A (en) * | 2020-10-16 | 2020-12-11 | 常州市艾斯康生物医药有限公司 | Pharmaceutical preparation for relieving and preventing allergic rhinitis |
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