CN102920652B - Propolis and chitosan periodontal slow-release thermo-sensitive in-situ gel and preparation method thereof - Google Patents
Propolis and chitosan periodontal slow-release thermo-sensitive in-situ gel and preparation method thereof Download PDFInfo
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Abstract
The invention provides propolis and chitosan periodontal slow-release thermo-sensitive in-situ gel and a preparation method thereof, and belongs to a preparation method for a periodontal treatment local slow-release medicament. The gel comprises a blank matrix thermo-sensitive in-situ gel of a chitosan acetic acid solution and an aqueous solution of beta-sodium glycerophosphate in a volume ratio; and an aqueous solution of propolis-coated hydroxypropyl beta-cyclodextrin in a mass ratio is used as a slow-release micro capsule of propolis/hydroxypropyl beta-cyclodextrin. The invention also provides the preparation method of the propolis and chitosan periodontal slow-release thermo-sensitive in-situ gel. Preferably, the chitosan of which the deacetylation degree is 90 percent and the molecular weight is 0.5 million Doyle is used as a medicament-carrying slow-release gel preparation. The gel forming temperature of the thermo-sensitive gel is 36 DEG C, the gel forming time is 65 seconds, the gel is released slowly and stably, the release degree reaches over 99 percent in 24 hours at the temperature of 37+/-0.5 DEG C, and a sudden release effect is avoided.
Description
Technical field
The invention belongs to the preparation method of periodontal disease therapeutic local sustained release medicine, particularly with temperature sensing in situ gel rubber, prepare the method for periodontal slow releasing pharmaceutical.
Background technology
In dental care field, all there is correlational study and the clinical report of propolis (propolis) both at home and abroad.Yong in 1997 etc. find by research, propolis has more obvious inhibitory action to the activity of oral cavity pathogen and enzyme, Bruschi in 2007 etc. have reported employing propolis slow releasing agent treatment periodontitis, preliminary study shows, this system has the effect of potential treatment periodontitis, be worth carrying out clinical evaluation, Jiang Lin in 2008 etc. have studied propolis and the impact of Propolis-ornidazole mixture on porphyromonas gingivalis growth, find that propolis has good inhibitory action to the growth of porphyromonas gingivalis, and can strengthen the antibacterial activity in vitro of ornidazole, simultaneously, can also reduce antibiotic use, thereby reduce the generation of drug side effect and bacterial drug resistance.Yet propolis is insoluble in water, topical mode is undesirable, has affected propolis in clinical application.
Situ-gel (in situ gel) refers to and can, with after solution state administration, at agents area, to environmental change response, occur immediately to change mutually forming non-chemically crosslinked semi-solid preparation.At present, the situ-gel system that can be used for human body mainly contains poloxamer gel systems, chitosan gel rubber system, alginate jelly system etc.Along with the development of macromolecular material, for the administration of situ-gel periodontal local sustained release provides good pharmaceutical carrier, make topical system highlight gradually its superiority as periodontal disease clinical medicine.
Chitosan is chitin deacetylase derivant, is the linear polysaccharide of a kind of natural biopolymer.At occurring in nature, chitosan extensively exists, and annual biosynthetic stock number, up to 10,000,000,000 tons, is the second largest living resources that are only second to Plant fiber on the earth.Chitosan in situ thermosensitive hydrogel (chitosan Thermosensitive in situ gel) can solution state administration and is occurred to change mutually at agents area, forms semi-solid preparation.Jothi (2009) etc. adopt chitosan thermosensitive hydrogel slow release
System parcel chlorine is applied to laboratory animal periodontitis, has obtained better therapeutic effect.Chitosan molecule structure chart is as follows:
Hydroxypropylβ-cyclodextrin is the hollow tube-shape molecule being formed by connecting with α-Isosorbide-5-Nitrae glycosidic bond by 7 glucose molecules.This molecule one end opening is larger, and the other end is less, and internal diameter is 6.0-6.5 à; Intramolecule is hydrophobicity, and outside is hydrophilic.Sizeable hydrophobic molecule easily enters in the middle hole of beta-schardinger dextrin-, form clathrate, by the molecule of enclose and beta-schardinger dextrin-, with hydrogen bond and Van der Waals force, interacted, the formed clathrate of hydrophobic molecule that makes to enter in the middle hole of beta-schardinger dextrin-has good stability.Special molecular structure based on beta-schardinger dextrin-, beta-schardinger dextrin-is often used to the preparation of medicine, essence slow-release microcapsule as wall material, to reach medicine and essence, slowly discharge and delay the object (Loftsson of oxidation deterioration, T.Pharmaceutical Sciences. 1996,85,1017-1024.).Hydroxypropylβ-cyclodextrin is as a kind of new function molecule, and to thermally-stabilised, to muscle and mucosa nonirritant almost, and without nephrotoxicity, dissolubility can be greater than 750g/L room temperature (20 ℃) is lower, the prescription by Europe and U.S.'s approval for oral and ejection preparation.
Summary of the invention
The object of the invention is to form the gelation time of chitosan thermosensitive hydrogel, plastic temperature are usingd as the blank substrate of thermosensitive hydrogel by experimental study molecular weight and deacetylation and other, simultaneously, using there is certain Mlc propolis extract as periodontal slow releasing pharmaceutical, study and provide the enclose material of the slow-release microcapsule of enclose propolis, provide and be suitable for the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber that propolis slow release discharges.
Another object of the present invention is to provide a kind of preparation technology of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
Propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of the present invention:
(1) take chitosan acetic acid solution that concentration is 2g/100mL and concentration as 125g/100mL sodium β-glycerophosphate aqueous solution by volume 5:2 mix, as blank substrate temperature sensing in situ gel rubber;
Wherein, the deacetylation of chitosan is 70%～95%, molecular weight is 100,000～900,000;
(2) take the hydroxypropylβ-cyclodextrin aqueous solution enclose propolis that concentration is 10g/100mL, the mass ratio between hydroxypropylβ-cyclodextrin aqueous solution and propolis is 1:2, as the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin propolis;
The minimum inhibitory concentration of described propolis is 1.25g/100mL;
(3) propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is mixed and is formed by the temperature sensing in situ gel rubber of the blank substrate of step (1) and the slow-release microcapsule of step (2) propolis/hydroxypropylβ-cyclodextrin propolis.
Described gel is further 90% for select deacetylation in step (1), the chitosan that molecular weight is 900,000.
The plastic temperature of described gel is 36 ℃, and gelation time is 65 seconds, in this thermosensitive hydrogel the slow-release microcapsule stripping of propolis/hydroxypropylβ-cyclodextrin slowly steady, propolis release in 37 ± 0.5 ℃, 24h reaches more than 99%.
The preparation method of one of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber:
(1) prepare blank substrate temperature sensing in situ gel rubber: the chitosan of getting deacetylation 70%～95%, molecular weight 100,000～900,000 with concentration 2g/100mL is dissolved in the acetic acid solution of 0.1mol/L, is stirred to dissolving, 4 ℃ of standing 1h; With concentration 125g/100mL, get sodium β-glycerophosphate and be dissolved in distilled water, be stirred to dissolving, 4 ℃ of standing 1h; Sodium β-glycerophosphate solution is dropwise added to chitosan solution, continue to stir 15min and fully mix, standing to bubble collapse;
(2) slow-release microcapsule of preparation propolis/hydroxypropylβ-cyclodextrin propolis: put 95% ethanol 8mL in beaker, add after 20g propolis in 60 ℃ of water-bath 45min, be dissolved as propolis solution; Getting hydroxypropylβ-cyclodextrin adds distilled water to dissolve to obtain the concentration cyclodextrin solution that is 10g/100mL; Propolis solution is splashed into cyclodextrin solution, under temperature 50 C, stir 120 minutes to obtain mixture, then mixture is passed through to 0.45 μ m microporous filter membrane filtering ethanol, obtain settled solution, standing, lyophilization 4h, obtain the slow-release microcapsule of yellowish, loose propolis/hydroxypropylβ-cyclodextrin propolis.
The minimum inhibitory concentration of described propolis is 1.25g/100mL.
(3) under agitation, the temperature sensing in situ gel rubber solution of the blank substrate of step (1) is added to step (2) propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule, when being evenly mixed to without obvious agglomerate, both survey pH, drip 0.1mol/L sodium hydroxide solution and regulate pH value to 7.0～7.3, the centrifugal 5min of 3000r/min, eliminate bubble and precipitation, clear liquor is propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
The preparation method of described gel, is characterized in that: it is 90% that step (1) is further selected deacetylation, the chitosan that molecular weight is 900,000.
The present invention has following positive effect:
1. the present invention is by the different deacetylations of research and different molecular weight, comprise deacetylation 70%, 80%, 90% and 95%, blank substrate chitosan temperature sensing in situ gel rubber plastic temperature and the gelation time of molecular weight 10 Wan Daoer, 30 Wan Daoer, 50 Wan Daoer, 70 Wan Daoer and 90 Wan Daoer, result shows: deacetylating degree of chitosan is larger on the impact of thermosensitive hydrogel plastic temperature, be that less its plastic temperature of deacetylation is higher, larger its plastic temperature of deacetylation is lower; And chitosan molecule amount is little on the plastic temperature impact of thermosensitive hydrogel.Therefore, it is 70%～95% that the present invention generally can select deacetylating degree of chitosan, and preferably deacetylation is that more than 90% chitosan is as the sustained-release gel preparation of carrying medicaments.
2 but for the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of the slow-release microcapsule of enclose propolis/hydroxypropylβ-cyclodextrin propolis, in the final measuring of the present invention, molecular weight is that the slow release effect of 90 Wan Daoer temperature sensing in situ gel rubber is better, thereby the general selectable chitosan molecule amount of the present invention is 100,000～900,000, the chitosan of preferred molecular weight 500,000 is as the sustained-release gel preparation of carrying medicaments.
3, the present invention investigates the preferred chitosan of institute by orthogonal test and single factor, formula and the technique of the blank substrate thermosensitive hydrogel of sodium β-glycerophosphate and the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin enclose, increased propolis water solublity, in end product, the plastic temperature of thermosensitive hydrogel is 36 ℃, gelation time is 65 seconds, and detect through vitro release experiment, propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule stripping of gel is slowly steady, propolis at 37 ± 0.5 ℃ in 24h release reach more than 99%, and drug release can not produce burst effect, reached the slow release effect of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
The invention belongs to the part in national natural science fund subsidy project (30960424) and Yunnan Province's social development plan of science and technology project (2007CA011), the result obtaining will be applied to production and the clinical practice of periodontal slow releasing preparation.
Accompanying drawing explanation
Fig. 1 is the intrinsic viscosity regression curve of chitosan.
Fig. 2 is the viscosity with temperature change curve of blank substrate temperature sensing in situ gel rubber.In figure, when lower temperature, gel viscosity coefficient is close.When temperature is increased near 35 ℃, gel viscosity increases sharply, by liquid state to semisolid gel conversion.When temperature exceeds 35 ℃, gel viscosity exceeds viscometer rotor maximum range.
Fig. 3 is that propolis/hydroxypropylβ-cyclodextrin proportioning is 1:2, and enclose temperature is 50 ℃, and the enclose time is propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule prepared by 120min, and this capsule is faint yellow, rarefaction.
Fig. 4 is that propolis is added into solution after blank substrate temperature sensing in situ gel rubber after by Polyethylene Glycol dissolution.This figure shows: in solution, separate out precipitation.
Fig. 5 is that propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds the situation in blank substrate temperature sensing in situ gel rubber to.This figure shows: even if capsule concentration raises, the viscosity of solution increases gradually, still has mobility; And capsule all can be dissolved in blank substrate temperature sensing in situ gel rubber completely, does not occur obvious insoluble matter.With respect to Fig. 4, its physical behavior has had larger improvement.
Fig. 6 is the plastic temperature correlation curve of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber and blank substrate temperature sensing in situ gel rubber.As Fig. 6, propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds the amount of blank substrate temperature sensing in situ gel rubber larger, and the temperature of the plastic of thermosensitive hydrogel is higher,
Fig. 7 is the gelation time correlation curve of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber and blank gel.As Fig. 7, propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds the amount of blank substrate temperature sensing in situ gel rubber larger, and the gelation time of thermosensitive hydrogel is longer.
Fig. 8 is the pH correlation curve of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber and blank gel.As Fig. 8, propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds the amount of blank substrate temperature sensing in situ gel rubber larger, and pH reduces rapidly along with the increase of slow-release microcapsule addition.
Fig. 9 be with spectrophotography at the absorbance of 415nm place mensuration propolis total flavones the time dependent curve of extracorporeal releasing quantity in 5 different molecular weight propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.The sample parallel assay of every curve three times, averages, and brings regression equation into.As shown in the figure, the propolis total flavones of 5 different molecular weight propolis-chitosan periodontal slow release temperature sensing in situ gel rubber discharges all can not produce burst effect, and about 20h approaches release completely after stripping.
Figure 10 is the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber under low temperature.
Figure 11 is the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of gelling.
Below in conjunction with specific embodiment, further illustrate the present invention, specific embodiment comprises but does not limit the scope of protection of the invention.
The specific embodiment
The preparation of one different deacetylation chitosans and mensuration
(1) preparation of the chitosan of different deacetylations
Get 1g deacetylation and be 90% chitosan and be dissolved in 60mL 12%(v/v) acetic acid solution, then add 60mL absolute methanol, and according to following table 1 ratio, add acetic anhydride, the response time,
The preparation of the chitosan of the different deacetylations of table 1
The potassium hydroxide-ethanol solution that adds 500mL 0.5mol/L, separates out white fiber shape material, with absolute ethanol washing, is neutral 2 times to solution, filters, and is dried to constant weight and obtains low deacetylation chitosan.By changing acetic anhydride addition, adjust the response time, prepared deacetylation and be 60%, 70%, 80% chitosan.Adopt iodine to make adsorption indicator titration measuring deacetylating degree of chitosan.
More than reaction in, chitosan is U.S. Amresco company, all the other reagent as methanol, acetic anhydride, ethanol, potassium hydroxide be analytical pure.
(2) different deacetylation chitosan solubility properties are measured
The deacetylation of preparation of the usining chitosan that is 60%, 70%, 80%, 90%, 95% is as subjects, and it is 2% chitosan solution that the acetic acid solutions that it is dissolved in respectively to 0.1mol/L becomes concentration, observes its dissolubility, and result is as table 2:
The different deacetylation chitosan of table 2 dissolubility
The mensuration of chitosan molecule amount: the sample of the chitosan molecule amount of take sign 100000 is the viscosity-average molecular weight that example is measured chitosan, sample solution passes through Ubbelohde viscometer, measuring known solvent fall time (T0) is 114.94 seconds, the fall time (in Table 2) that records respectively variable concentrations solution, calculates η
sP/ C, with η
sP/ C-C is figure (seeing Fig. 1), can obtain intrinsic viscosity [η]=887.42, by [η]=KM
athe viscosity-average molecular weight M=134682 that calculates chitosan conforms to substantially with 100000 molecular weight that producer indicates.
Table 3 fall time and intrinsic viscosity relation
Dissolubility to different deacetylation chitosans is studied, result demonstration, and the chitosan major part of 60% deacetylation can not be dissolved in acetic acid, and its solution viscosity is larger.The present invention selects the chitosan of 70%, 80%, 90%, 95% deacetylation to carry out follow-up test.
Two chitosans and sodium β-glycerophosphate combine preparation and the test of blank substrate temperature sensing in situ gel rubber
(1) preparation of blank substrate temperature sensing in situ gel rubber
According to the form below 4,5,6, the chitosan of getting respectively listed amount is dissolved in the acetic acid solution of 0.1mol/L, is placed in the lasting 1h of stirring on magnetic stirring apparatus and fully dissolves, and 4 ℃ of standing 1h are standby.Press table 4,5,6, the sodium β-glycerophosphate of getting respectively listed amount is dissolved in distilled water, is placed on magnetic stirring apparatus, to stir 15min and dissolve completely, and 4 ℃ of standing 1h are standby.In table 4,5,6 ratio, under stirring, sodium β-glycerophosphate solution is dropwise added to chitosan solution, continue to stir 15min and fully mix, be statically placed in refrigerator and remove bubble, standby.
(2) plastic temperature and the gelation time of the blank substrate temperature sensing in situ gel rubber of different deacetylations
Adopt test tube anastrophe, the water-bath that the blank substrate temperature sensing in situ gel rubber of different deacetylations is placed in to different temperatures, measures respectively plastic temperature, the gelation time of each sample.Each sample parallel assay 3 times, result is got its meansigma methods.
As shown in table 3, when chitosan molecule amount and concentration, sodium β-glycerophosphate concentration one regularly, along with the increase of deacetylating degree of chitosan, the plastic temperature of blank substrate temperature sensing in situ gel rubber reduces gradually.In experimental result: the gelation time of the blank substrate temperature sensing in situ gel rubber of 70% deacetylation is the longest, approximately 305 seconds ability plastics of 63 ℃ of water-baths, and the gelation time of 95% deacetylation is the shortest, 32 ℃ of water-baths approximately 65 seconds just can plastic; And the blank substrate temperature sensing in situ gel rubber of 95% deacetylation is preserved under environment and is formed voluntarily gel after 48 hours at 4 ℃.Thereby the chitosan of higher deacetylation can reduce the plastic temperature of blank substrate temperature sensing in situ gel rubber, shortens its gelation time.
Table 4 changes with chitosan thermosensitive hydrogel plastic temperature, the gelation time of deacetylation
(3) chitosan of different molecular weight affects gel formation
Adopt test tube anastrophe, the blank substrate temperature sensing in situ gel rubber of different molecular weight is placed in to the water-bath of 37 ± 0.5 ℃, measure respectively plastic temperature, the gelation time of each sample.Each sample parallel assay 3 times, result is got its meansigma methods.
As shown in table 4, when deacetylating degree of chitosan and concentration, sodium β-glycerophosphate concentration one regularly, along with the increase of chitosan molecule amount, the plastic temperature of blank substrate temperature sensing in situ gel rubber is without too large change.In this experiment, choosing chitosan molecule amount in 100,000～900,000 scopes, is that a gradient increases progressively by every 200,000, amounts to 5 different molecular weights.Bath temperature scope is at 15 ℃～40 ℃, and the blank substrate temperature sensing in situ gel rubber of variant molecular weight solution all can occur gelling phenomenon, result demonstration, and the molecular weight of chitosan does not have a significant effect to the plastic temperature of blank substrate temperature sensing in situ gel rubber.
The impact of the blank substrate temperature sensing in situ gel rubber of table 5 different molecular weight on plastic temperature
(4) L of blank substrate temperature sensing in situ gel rubber
9(3
4) orthogonal test
The proportioning of take between deacetylation 90%, molecular weight 50 Wan Daoer chitosan solution concentration, sodium β-glycerophosphate solution concentration and two solution is investigation factor, respectively get 3 levels, the plastic temperature of temperature sensing in situ gel rubber of blank substrate of take is evaluation index, carries out Three factors-levels L
9(3
4) orthogonal test, design optimization prescription substrate.Test empirical factor level is as table 6, and orthogonal test table is as table 7.
Table 6 empirical factor level
Table 7 orthogonal experiments
Table 8 analysis of variance table
F
0.05(2,2)=19.00,F
0.1(2，2)=99.0
By being A3B1C1 to the optimum process condition screening after above-mentioned optimal design, chitosan and sodium β-glycerophosphate ratio are 1:1, and sodium β-glycerophosphate concentration is 50%, and chitosan concentration is 2%.Plastic temperature and the gelation time of measuring these process conditions are: 35 ℃ of gelation times 70 seconds, 37 ℃ of gelation times 110 seconds.Using the foundation of this experimental result as final experiment.
Keep chitosan and sodium β-glycerophosphate consumption constant, the water content of take in solution is empirical factor, gets three levels, investigates its impact on blank substrate temperature sensing in situ gel rubber gelation time, plastic temperature, empirical factor level and the results are shown in Table 9, table 10.
Table 9 empirical factor level
The single factor of table 10 is investigated result
As shown in Table 9, in the situation that all the other conditions are constant, the consumption that reduces water in solution can effectively reduce plastic temperature and the gelation time of blank substrate temperature sensing in situ gel rubber.In prepared gel solution, have a small amount of macroscopic precipitate, after centrifugal removal precipitate, the plastic temperature of gel and gelation time are without obvious change.
(5) blank substrate temperature sensing in situ gel rubber rheology is investigated
The apparent viscosity of blank substrate temperature-sensitive situ-gel system while being determined at different temperatures with rotary viscosimeter.Get gel 10mL to be measured and be placed in the sample cell that internal diameter is 20mm, with the speed of 1～2 ℃/min, be slowly warmed up to 40 ℃, select the suspension rotor of suitable range to immerse sample to scale, measure the viscosity under different temperatures.Since 15 ℃, a viscosity number of 1 ℃ of record of the every rising of temperature, to exceeding viscometer rotor range.Gel sample parallel assay in each sample cell 3 times, results averaged, prepares gel apparent viscosity curve as Fig. 2.
Preparation and the test of the slow-release microcapsule of three propolis/hydroxypropylβ-cyclodextrin propolis
(1) prepare the reason of the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin propolis
Propolis water solublity is poor, and oxidizable rotten, propolis dissolubility in chitosan thermosensitive hydrogel system is too low, causes drug effect to be difficult to performance.Slow-release microcapsule technique is propolis to be carried out to dissolution process its dissolubility of increase, makes gel slow release Mlc reposefully.In this experimentation, also attempt adding the cosolvents such as carboxymethyl cellulose, Polyethylene Glycol and promoted the dissolubility of propolis in blank substrate temperature sensing in situ gel rubber, all do not obtain good effect.
(2) with L
9(3
4) Orthogonal Experiment and Design carries out the preparation of the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin propolis
Take propolis and hydroxypropylβ-cyclodextrin proportioning, temperature, mixing time is factor, respectively gets 3 levels, and empirical factor level is in Table 10.The propolis content of clathrate of take is evaluation index, carries out L
9(3
4) orthogonal test.Orthogonal Experiment and Design and the results are shown in Table 11.
Press L
9(3
4) Orthogonal Experiment and Design, get hydroxypropylβ-cyclodextrin and add appropriate distilled water, after dissolved solution, hydroxypropylβ-cyclodextrin solution is placed on agitator; Get propolis and add 95% ethanol 8mL in beaker, 60 ℃ of about 45min of water-bath Ultrasonic Heating dissolve, and propolis solution is dropwise added in agitator cyclodextrin solution.Afterwards, filtering ethanol, obtains settled solution by solution by 0.45 μ m filtering with microporous membrane, and standing, freeze drying box is dried 4h, obtains the slow-release microcapsule of yellowish, loose propolis/hydroxypropylβ-cyclodextrin propolis.Get capsule 0.1g and put in beaker, add 50% ethanol 10mL, in the 25 ℃ of ultrasonic concussion heating of water-bath 45min, make it to dissolve.With reference to GB2007, with spectrophotography, at 415nm place mensuration absorbance, according to regression equation, obtain the content of propolis in the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin propolis.
Table 11 empirical factor level
Table 12 Orthogonal Experiment and Design and result
Table 13 variance analysis
F
0.05(2,2)=19.00,F
0.1(2，2)=99.0
From the results of analysis of variance, shown, factor A has the impact of highly significant on experimental result, and factor B and factor C are not obvious on the impact of experimental result.And from intuitive analysis, factor A is the most remarkable on the amount impact of hydroxypropylβ-cyclodextrin, K
1a> K
2a> K
3atherefore, factor A selection level 1, propolis and hydroxypropylβ-cyclodextrin ratio are 1:2; Factor B also has certain influence, K to experimental result
3b> K
2b> K
1btherefore, factor B selection level 3, enclose temperature is 50 ℃; Factor C is not significant factors, K
3c> K
2c> K
1c, therefore select C3 level, the enclose time is 120 minutes.Can obtain thus, optimum process condition is A1B3C3, and propolis and beta-schardinger dextrin-proportioning are 1:2, temperature 50 C, stirs 120 minutes, with the prescription of this condition, propolis is carried out to enclose, can increase the water solublity of slow-release microcapsule, for clinical periodontal application propolis provides experimental basis.
Preparation and the detecting for slowly-releasing property thereof of four propolis-chitosan periodontal slow release temperature sensing in situ gel rubber
(1) gelling property of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is measured
By the chitosan of the deacetylation of concentration 2g/100mL 90%, the sodium β-glycerophosphate of concentration 50g/100mL by volume 1:1 mixes as blank matrix gel, then this blank matrix gel and the slow-release microcapsule that contains the propolis/hydroxypropylβ-cyclodextrin of 5g/100mL, 10g/100mL, tri-kinds of concentration of 20g/100mL are formed to propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
Detect the plastic temperature of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber, gelation time and pH, result is as Fig. 6,7,8.As figure, the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin adds the amount of blank matrix gel larger, and the temperature of the plastic of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is higher, simultaneously, gelation time extends, and pH reduces rapidly along with the increase of slow-release microcapsule addition.
Experiment is found: during propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule concentration 5g/100mL, add the alkaline solution chitosan thermosensitive hydrogel pH that can raise, its plastic temperature adjustment is returned to 37 ℃; When propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule concentration increases to 10g/100mL, its plastic temperature is just difficult to adjust back 37 ℃.Experiment is also found: in the situation that keeping chitosan and sodium β-glycerophosphate consumption constant, reduce the content of water in thermosensitive hydrogel system, can effectively reduce plastic temperature and the gelation time of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
Get the propolis that minimum inhibitory concentration is 1.25g/100mL, concentration 10g/100mL propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule is added in the temperature sensing in situ gel rubber of blank substrate of three different prescriptions that single factor investigates, investigate content as table 14.
The impact of table 14 water content on gel-forming property
Drip the sodium hydroxide solution of 0.1mol/L to pH value 7.13 left and right, investigate its plastic temperature and gelation time, result is as table 15:
The single factor of table 15 is investigated result
In chitosan thermosensitive hydrogel, add as shown in Table 15 after propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule of recipe quantity 10g/100mL, by reducing the content of water in solution, the plastic temperature of rising can be recalled near 37 ℃ of body temperatures, and can shorten its gelation time.
(2) preparation of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber
According to the requirement of clinical oral local application, the chitosan of choice experiment A deacetylation 90%, molecular weight 500,000 and sodium β-glycerophosphate aqueous solution, wherein, chitosan acetic acid solution concentration is 2g/100mL, sodium β-glycerophosphate concentration of aqueous solution is 125g/100mL, chitosan acetic acid solution volume and sodium β-glycerophosphate aqueous solution volume ratio are 5:2, as the temperature sensing in situ gel rubber of blank substrate.Stir this gel solution, propolis/hydroxypropylβ-cyclodextrin the slow-release microcapsule that adds concentration 10g/100mL, it is uniformly dispersed, during to the obvious agglomerate of nothing, measure pH, drip a small amount of 0.1mol/L sodium hydroxide solution and regulate pH value to 7.0～7.3, the centrifugal 5min of 3000r/min, eliminate bubble and precipitation, settled solution is propolis-chitosan periodontal slow release temperature sensing in situ gel rubber, is placed in that 4 ℃ of environment are preserved, bubble to be eliminated is standby.
(3) investigation of the In Vitro Dissolution character of propolis flavone
By (2) identical method, preparing deacetylation is 90%, molecular weight is 100,000,300,000,500,000,700,000,900,000 propolis-chitosan periodontal slow release temperature sensing in situ gel rubber, get respectively each 2mL and put into the little cillin bottle of 20mm, after bubble is eliminated completely, in 37 ± 0.5 ℃ of constant temperature oscillators, preheating 10 min make it to form gel, the artificial saliva 2mL that adds respectively again 37 ± 0.5 ℃ is release medium, frequency is 50r/min, amplitude is 20mm, respectively at after vibration in 2,4,6,8,10,12,14,16,20,24h sampling.At the appointed time artificial saliva is all poured out, supplement immediately 2mL artificial saliva.Sample filters with 0.45 μ m microporous filter membrane (water system), gets subsequent filtrate 1.5mL and lets cool, and by 2 times of artificial saliva dilution, shakes up.The extracorporeal releasing quantity time dependent curve of the absorbance of measuring propolis total flavones with spectrophotography at 415nm place in 5 different molecular weight propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.The sample parallel assay of every curve three times, averages, and brings regression equation into.
As shown in Figure 9, the propolis total flavones of 5 different molecular weight chitosan thermosensitive hydrogel discharges all can not produce burst effect, and about 20h approaches release completely after stripping.And 1,2 two sample, 3,4 two sample dissolution rates are more consistent, and the dissolution rate of 1,2 two sample is fast compared with 3,4 two samples; Other 4 samples are slower relatively for the dissolution rate of sample 5, and the dissolution rate of more other 4 samples is more even.
Above artificial saliva adopts the one-tenth assignment system of Fusayama Meyer type artificial saliva: distilled water 1000mL; Urea (carbamide) 1.0g/L; NaC1 0.4g/L; KC1 0.4g/L; NaH2P042H20 0.78g/L; Na2S2H20 0.005 g/L; CaC122H2O 0.795g/L.37(± 0.5) ℃ probe temperature:.
Sun Jian, Yin Zhihua, fourth Zhong Juan. the experiment in vitro [J] of poloxamer thermosensitive hydrogel blend cyclodextrin slow release propolis. the Academic Journal of Kunming Medical College, 2011,32 (12): 14-17.
Sun Jian, fourth Zhong Juan. the repairing and treating method of patients with periodontitis. the Academic Journal of Kunming Medical College, 2011,32 (2B): 169-171.
Yin Zhihua, Sun Jian, fourth Zhong Juan. in propolis
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Claims (4)
1. propolis-chitosan periodontal slow release temperature sensing in situ gel rubber, is characterized in that this gel:
(1) take chitosan acetic acid solution that concentration is 2g/100mL and concentration as 125g/100mL sodium β-glycerophosphate aqueous solution by volume 5:2 mix, as blank substrate temperature sensing in situ gel rubber;
Wherein, the deacetylation of chitosan is 70%～95%, molecular weight is 100,000～900,000;
(2) take the hydroxypropylβ-cyclodextrin aqueous solution enclose propolis that concentration is 10g/100mL, the mass ratio between hydroxypropylβ-cyclodextrin aqueous solution and propolis is 1:2, as the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin;
The minimum inhibitory concentration of described propolis is 1.25g/100mL;
(3) propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is mixed and is formed by the temperature sensing in situ gel rubber of the blank substrate of step (1) and the slow-release microcapsule of step (2) propolis/hydroxypropylβ-cyclodextrin;
The plastic temperature of above-mentioned steps (3) propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is 36 ℃, gelation time is 65 seconds, in this thermosensitive hydrogel, the slow-release microcapsule stripping of propolis/hydroxypropylβ-cyclodextrin is slowly steady, and propolis release in 37 ± 0.5 ℃, 24h reaches more than 99%.
2. propolis-chitosan periodontal slow release temperature sensing in situ gel rubber according to claim 1, is characterized in that: it is 90% that step (1) is further selected deacetylation, the chitosan that molecular weight is 900,000.
3. the preparation method of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber as claimed in claim 1 or 2, is characterized in that:
(1) prepare blank substrate temperature sensing in situ gel rubber: the chitosan of getting deacetylation 70%～95%, molecular weight 100,000～900,000 with concentration 2g/100mL is dissolved in the acetic acid solution of 0.1mol/L, is stirred to dissolving, 4 ℃ of standing 1h; With concentration 125g/100mL, get sodium β-glycerophosphate and be dissolved in distilled water, be stirred to dissolving, 4 ℃ of standing 1h; Sodium β-glycerophosphate solution is dropwise added to chitosan solution, continue to stir 15min and fully mix, standing to bubble collapse;
(2) slow-release microcapsule of preparation propolis/hydroxypropylβ-cyclodextrin: put 95% ethanol 8mL in beaker, add after 20g propolis in 60 ℃ of water-bath 45min, be dissolved as propolis solution; Getting hydroxypropylβ-cyclodextrin adds distilled water to dissolve to obtain the concentration cyclodextrin solution that is 10g/100mL; Propolis solution is splashed into cyclodextrin solution, under temperature 50 C, stir 120 minutes to obtain mixture, then mixture is passed through to 0.45 micron of microporous filter membrane filtering ethanol, obtain settled solution, standing, lyophilization 4h, obtain the slow-release microcapsule of yellowish, loose propolis/hydroxypropylβ-cyclodextrin;
The minimum inhibitory concentration of above-mentioned propolis is 1.25g/100mL;
(3) under agitation, the temperature sensing in situ gel rubber solution of the blank substrate of step (1) is added to step (2) propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule, when being evenly mixed to without obvious agglomerate, both survey pH, drip 0.1mol/L sodium hydroxide solution and regulate pH value to 7.0～7.3, the centrifugal 5min of 3000r/min, eliminate bubble and precipitation, clear liquor is propolis-chitosan periodontal slow release temperature sensing in situ gel rubber.
4. the preparation method of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber according to claim 3, is characterized in that: step (1) is further selected the chitosan that deacetylation is 90%, molecular weight is 900,000.
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| FR3029790B1 (en) * | 2014-12-12 | 2017-01-13 | Synolyne Pharma | CHITOSAN HYDROGEL MICROBELL |
| CN107281540A (en) * | 2016-04-11 | 2017-10-24 | 李永生 | Treat the Nano Silver temperature-sensitive hydrogel and preparation method of burn and scald |
| CN106177989A (en) * | 2016-09-21 | 2016-12-07 | 青岛农业大学 | A kind of tilmicosin clathrate chitosan thermosensitive hydrogel and preparation method thereof |
| CN108310006A (en) * | 2017-08-14 | 2018-07-24 | 南京医科大学附属口腔医院 | A kind of pharmaceutical composition and its preparation method and application of prevention dental caries |
| WO2021015703A2 (en) * | 2019-07-24 | 2021-01-28 | Firat Universitesi Rektorlugu | Nanopropolis synthesis method with healing effect against side effects of cancer drug cisplatin |
| CN110664725B (en) * | 2019-11-22 | 2021-11-30 | 北京中蜜科技发展有限公司 | Preparation method of emulsion containing nanometer propolis extract and emulsion prepared thereby |
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| "泊洛沙姆温敏凝胶共混环糊精缓释蜂胶的体外实验";孙剑等;《昆明医学院学报》;20111231;第32卷(第12期);第14-17页 * |
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