CN102920652A - 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 prepare the method for periodontal slow releasing pharmaceutical with temperature sensing in situ gel rubber.
Background technology
In the dental care field, correlational study and the clinical report of propolis (propolis) arranged all 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 Propolis-ornidazole mixture to the porphyromonas gingivalis affects on the growth, find that propolis has preferably 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, and the topical mode is undesirable, has affected propolis in clinical application.
Situ-gel (in situ gel) refers to and can with after the solution state administration, to environmental change response, occur immediately to change mutually forming non-chemically crosslinked semi-solid preparation at agents area.At present, the situ-gel system that can be used for human body mainly contains the 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 the topical system highlight gradually its superiority as the periodontal disease clinical medicine.
Chitosan is the 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 is the second largest living resources that are only second to the Plant fiber on the earth up to 10,000,000,000 tons.Chitosan in situ thermosensitive hydrogel (chitosan Thermosensitive in situ gel) can the solution state administration and is occured to change mutually at agents area, forms semi-solid preparation.Jothi (2009) etc. adopt chitosan thermosensitive hydrogel slow release
System's parcel chlorine is applied to the laboratory animal periodontitis, has obtained better therapeutic effect.The chitosan molecule structure chart is as follows:
Hydroxypropylβ-cyclodextrin is the hollow tube-shape molecule that is 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 the outside is hydrophilic.Sizeable hydrophobic molecule easily enters in the middle hole of beta-schardinger dextrin-, form clathrate, interacted with hydrogen bond and Van der Waals force by the molecule of enclose and beta-schardinger dextrin-, make the formed clathrate of hydrophobic molecule in the middle hole that enters beta-schardinger dextrin-have preferably stability.Special molecular structure based on beta-schardinger dextrin-, beta-schardinger dextrin-often is used to the preparation of medicine, essence slow-release microcapsule as the wall material, slowly discharge and delay the purpose (Loftsson of oxidation deterioration to reach medicine and essence, T.Pharmaceutical Sciences. 1996,85,1017-1024.).Hydroxypropylβ-cyclodextrin is as a kind of new function molecule, to thermally-stabilised, to muscle and mucosa nonirritant almost, and without nephrotoxicity, dissolubility room temperature (20 ℃) is lower can be greater than 750g/L, be used for the prescription of oral and ejection preparation by Europe and U.S.'s approval.
Summary of the invention
The objective of the invention is to form gelation time to the chitosan thermosensitive hydrogel, becomes the glue temperature with as thermosensitive hydrogel blank substrate by experimental study molecular weight and deacetylation and other, simultaneously, with propolis extract with certain Mlc as the periodontal slow releasing pharmaceutical, study and provide the enclose material of the slow-release microcapsule of enclose propolis, provide to be suitable for the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber that the propolis slow release discharges.
Another purpose of the present invention provides 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) the chitosan acetic acid solution take concentration as 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) the hydroxypropylβ-cyclodextrin aqueous solution enclose propolis take concentration as 10g/100mL, the mass ratio between hydroxypropylβ-cyclodextrin aqueous solution and the 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 forms with step (2) propolis/slow-release microcapsule of hydroxypropylβ-cyclodextrin propolis by the temperature sensing in situ gel rubber of the blank substrate of step (1).
Described gel is 90% for select deacetylation in step (1) further, and molecular weight is 900,000 chitosan.
The one-tenth glue 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) the blank substrate temperature sensing in situ gel rubber of preparation: 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, and 4 ℃ leave standstill 1h; Get sodium β-glycerophosphate with concentration 125g/100mL and be dissolved in the distilled water, be stirred to dissolving, 4 ℃ leave standstill 1h; Sodium β-glycerophosphate solution is dropwise added chitosan solution, continue to stir 15min and fully mix, leave standstill to bubble collapse;
(2) slow-release microcapsule of preparation propolis/hydroxypropylβ-cyclodextrin propolis: put 95% ethanol 8mL in beaker, in 60 ℃ of water-bath 45min, be dissolved as propolis solution behind the adding 20g propolis; Get hydroxypropylβ-cyclodextrin adding dissolved in distilled water and get the cyclodextrin solution that concentration is 10g/100mL; Propolis solution is splashed into cyclodextrin solution, under temperature 50 C, stir 120 minutes to get mixture, again mixture is passed through 0.45 μ m microporous filter membrane filtering ethanol, obtain settled solution, leave standstill, lyophilization 4h, get 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 step (2) propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule, when evenly being mixed to without obvious agglomerate, both survey pH, drip the 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, and molecular weight is 900,000 chitosan.
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%, the blank substrate chitosan temperature sensing in situ gel rubber of molecular weight 10 Wan Daoer, 30 Wan Daoer, 50 Wan Daoer, 70 Wan Daoer and 90 Wan Daoer becomes glue temperature and gelation time, the result shows: deacetylating degree of chitosan becomes glue temperature effect larger to thermosensitive hydrogel, be that less its of deacetylation becomes the glue temperature higher, larger its of deacetylation becomes the glue temperature lower; And the chitosan molecule amount is little to the one-tenth glue temperature effect of thermosensitive hydrogel.Therefore, it is 70%～95% that the present invention generally can select deacetylating degree of chitosan, and preferred deacetylation is that chitosan more than 90% 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, and 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, prescription and the technique of the blank substrate thermosensitive hydrogel of sodium β-glycerophosphate and the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin enclose, increased the propolis water solublity, in end product, the one-tenth glue temperature of thermosensitive hydrogel is 36 ℃, gelation time is 65 seconds, and detect through the vitro release experiment, the propolis of gel/hydroxypropylβ-cyclodextrin slow-release microcapsule stripping is slowly steady, propolis release in 37 ± 0.5 ℃ of lower 24h reaches more than 99%, and drug release can not produce burst effect, has 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 the Yunnan Province's social development plan of science and technology project (2007CA011), the result who obtains will be applied to production and the clinical practice of periodontal slow releasing preparation.
Description of drawings
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.Among the figure, when lower temperature, the gel viscosity coefficient is close.When temperature is increased near 35 ℃, gel viscosity increases sharply, by liquid state to the semisolid gel conversion.When temperature exceeded 35 ℃, gel viscosity exceeded viscometer rotor maximum range.
Fig. 3 is that propolis/hydroxypropylβ-cyclodextrin proportioning is 1:2, and the enclose temperature is 50 ℃, and the enclose time is the propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule of 120min preparation, and this capsule is faint yellow, rarefaction.
Fig. 4 is added into solution behind the blank substrate temperature sensing in situ gel rubber after to be propolis short molten by Polyethylene Glycol.This figure shows: separate out precipitation in the solution.
Fig. 5 is that propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds the situation in the blank substrate temperature sensing in situ gel rubber to.This figure shows: even capsule concentration raises, the viscosity of solution increases gradually, still has flowability; And capsule all can be dissolved in the blank substrate temperature sensing in situ gel rubber fully, obvious insoluble matter do not occur.With respect to Fig. 4, its physical behavior has had larger improvement.
Fig. 6 be propolis-chitosan periodontal slow release temperature sensing in situ gel rubber with blank substrate temperature sensing in situ gel rubber become glue temperature correlation curve.Such as Fig. 6, the amount that propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds blank substrate temperature sensing in situ gel rubber is larger, and the temperature of the one-tenth glue 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.Such as Fig. 7, the amount that propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds blank substrate temperature sensing in situ gel rubber is 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.Such as Fig. 8, the amount that propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule adds blank substrate temperature sensing in situ gel rubber is larger, and pH reduces rapidly along with the increase of slow-release microcapsule addition.
Fig. 9 is the extracorporeal releasing quantity time dependent curve of absorbance in 5 different molecular weight propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of measuring propolis total flavones with spectrophotography at the 415nm place.The sample parallel assay of every curve three times is averaged, 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, discharges fully and approximately 20h is approaching after stripping.
Figure 10 is the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber under the low temperature.
Figure 11 is the propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of gelling.
Further specify the present invention below in conjunction with specific embodiment, 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 the 1g deacetylation and be 90% chitosan and be dissolved in 60mL 12%(v/v) acetic acid solution, add again the 60mL absolute methanol, add acetic anhydride according to following table 1 ratio, the response time,
The preparation of the chitosan of the different deacetylations of table 1
Add the potassium hydroxide-ethanol solution of 500mL 0.5mol/L, separate out white fiber shape material, be neutral with absolute ethanol washing 2 times to solution, filtration is dried to constant weight and obtains the low deacetylation chitosan.By changing the 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.
In the above reaction, chitosan is U.S. Amresco company, and all the other reagent such as methanol, acetic anhydride, ethanol, potassium hydroxide are analytical pure.
(2) different deacetylation chitosan solubility properties are measured
As subjects, it is 2% chitosan solution that its acetic acid solutions that is dissolved in respectively 0.1mol/L is become concentration, observes its dissolubility, result such as table 2 take the chitosan of deacetylation as 60%, 70%, 80%, 90%, 95% of preparation:
The different deacetylation chitosan of table 2 dissolubility
The mensuration of chitosan molecule amount: the viscosity-average molecular weight of measuring chitosan take the sample of chitosan molecule amount sign 100000 as example, sample solution passes through Ubbelohde viscometer, measuring as can be known, solvent fall time (T0) is 114.94 seconds, record respectively the fall time (seeing Table 2) of variable concentrations solution, calculate η
SP/ C is with η
SP/ C-C is figure (seeing Fig. 1), can get 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's 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 make up 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, places on the magnetic stirring apparatus to continue to stir 1h and fully dissolve, and 4 ℃ to leave standstill 1h for subsequent use.Press table 4,5,6, the sodium β-glycerophosphate of getting respectively listed amount is dissolved in the distilled water, places on the magnetic stirring apparatus to stir 15min and dissolve fully, and 4 ℃ to leave standstill 1h for subsequent use.In table 4,5,6 ratio, stir down, sodium β-glycerophosphate solution is dropwise added chitosan solution, continue to stir 15min and fully mix, be statically placed in and remove bubble in the refrigerator, for subsequent use.
(2) one-tenth glue temperature and the gelation time of the blank substrate temperature sensing in situ gel rubber of different deacetylations
Adopt the test tube anastrophe, with the water-bath that the blank substrate temperature sensing in situ gel rubber of different deacetylations places different temperatures, measure respectively one-tenth glue temperature, the gelation time of each sample.Each sample parallel assay 3 times, the result gets 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 one-tenth glue temperature of blank substrate temperature sensing in situ gel rubber reduces gradually.In the experimental result: the gelation time of the blank substrate temperature sensing in situ gel rubber of 70% deacetylation is the longest, and 63 ℃ of water-baths approximately 305 seconds ability become glue, and the gelation time of 95% deacetylation is the shortest, and 32 ℃ of water-baths approximately just can become glue in 65 seconds; And the blank substrate temperature sensing in situ gel rubber of 95% deacetylation is preserved under the environment at 4 ℃ and is formed voluntarily gel after 48 hours.Thereby the chitosan of higher deacetylation can reduce the one-tenth glue temperature of blank substrate temperature sensing in situ gel rubber, shortens its gelation time.
Table 4 becomes glue temperature, gelation time to change with the chitosan thermosensitive hydrogel of deacetylation
(3) chitosan of different molecular weight affects gel formation
Adopt the test tube anastrophe, the blank substrate temperature sensing in situ gel rubber of different molecular weight is placed 37 ± 0.5 ℃ water-bath, measure respectively one-tenth glue temperature, the gelation time of each sample.Each sample parallel assay 3 times, the result gets 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 one-tenth glue temperature of blank substrate temperature sensing in situ gel rubber is without too large change.Choosing the chitosan molecule amount in this experiment in 100,000～900,000 scopes, is that a gradient increases progressively by per 200,000, amounts to 5 different molecular weights.The bath temperature scope is at 15 ℃～40 ℃, and the blank substrate temperature sensing in situ gel rubber of variant molecular weight solution all the gelling phenomenon can occur, result's demonstration, and the molecular weight of chitosan is on the not obviously impact of one-tenth glue temperature of blank substrate temperature sensing in situ gel rubber.
The blank substrate temperature sensing in situ gel rubber of table 5 different molecular weight is on becoming the impact of glue temperature
(4) L of blank substrate temperature sensing in situ gel rubber
9(3
4) orthogonal test
Take the proportioning between deacetylation 90%, molecular weight 50 Wan Daoer chitosan solution concentration, sodium β-glycerophosphate solution concentration and two solution as the investigation factor, respectively get 3 levels, take the one-tenth glue temperature of the temperature sensing in situ gel rubber of blank substrate as evaluation index, carry out Three factors-levels L
9(3
4) orthogonal test, design optimization prescription substrate.Test empirical factor level such as table 6, orthogonal test table such 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 that screens after the above-mentioned optimal design, namely chitosan and sodium β-glycerophosphate ratio are 1:1, and sodium β-glycerophosphate concentration is 50%, and chitosan concentration is 2%.One-tenth glue temperature and the gelation time of measuring these process conditions are: 35 ℃ of gelation times 70 seconds, 37 ℃ of gelation times 110 seconds.With the foundation of this experimental result as final experiment.
Keep chitosan and sodium β-glycerophosphate consumption constant, the water content in the solution is got three levels as empirical factor, investigate its on blank substrate temperature sensing in situ gel rubber gelation time, become the impact of glue temperature, the 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 the result
As shown in Table 9, in the situation that all the other conditions are constant, the consumption that reduces water in the solution can effectively reduce one-tenth glue temperature and the gelation time of blank substrate temperature sensing in situ gel rubber.A small amount of macroscopic precipitate is arranged in the prepared gel solution, and behind centrifugal removal precipitate, the one-tenth glue 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 when being determined at different temperatures with rotary viscosimeter.Getting gel 10mL to be measured, to place internal diameter be the sample cell of 20mm, slowly is warmed up to 40 ℃ with the speed of 1～2 ℃/min, selects the suspension rotor of suitable range to immerse sample to scale, measures the viscosity under the different temperatures.Since 15 ℃, viscosity number of 1 ℃ of record of the every rising of temperature is to exceeding viscometer rotor range.Gel sample parallel assay in each sample cell 3 times, results averaged, preparation gel apparent viscosity curve such as Fig. 2.
Preparation and the test of the slow-release microcapsule of three propolis/hydroxypropylβ-cyclodextrin propolis
(1) reason of the slow-release microcapsule of preparation propolis/hydroxypropylβ-cyclodextrin propolis
The propolis water solublity is relatively poor, easy oxidation deterioration, and propolis dissolubility in chitosan thermosensitive hydrogel system is excessively low, causes drug effect to be difficult to performance.Slow-release microcapsule technique is propolis to be urged molten processing increase its dissolubility, makes reposefully slow release Mlc of gel.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 preferably 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 as factor, respectively get 3 levels, the empirical factor level sees Table 10.Take the propolis content of clathrate as evaluation index, carry 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 an amount of distilled water, after the dissolved solution, hydroxypropylβ-cyclodextrin solution is placed on the agitator; Get propolis and add 95% ethanol 8mL in beaker, 60 ℃ of approximately 45min dissolvings of water-bath Ultrasonic Heating dropwise add propolis solution in the agitator cyclodextrin solution.Afterwards, filtering ethanol obtains settled solution with solution by 0.45 μ m filtering with microporous membrane, leaves standstill, and the dry 4h of freeze drying box obtains slow-release microcapsule yellowish, the propolis that loosens/hydroxypropylβ-cyclodextrin propolis.Get capsule 0.1g and put in the beaker, add 50% ethanol 10mL, make it dissolving in the 25 ℃ of ultrasonic concussion heating of water-bath 45min.With reference to GB2007, at 415nm place mensuration absorbance, obtain the content of propolis in the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin propolis with spectrophotography according to regression equation.
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
Shown as can be known by the results of analysis of variance, 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 by intuitive analysis as can be known, factor A is the most remarkable on the amount impact of hydroxypropylβ-cyclodextrin, K
1a＞K
2a＞K
3aTherefore, factor A selection level 1, namely 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, namely the enclose temperature is 50 ℃; Factor C is not significant factors, K
3c＞K
2c＞K
1c, therefore select the C3 level, namely the enclose time is 120 minutes.Can get thus, optimum process condition is A1B3C3, and namely propolis and beta-schardinger dextrin-proportioning are 1:2, temperature 50 C stirred 120 minutes, with the prescription of this condition propolis was carried out enclose, can increase the water solublity of slow-release microcapsule, use propolis for clinical periodontal experimental basis is provided.
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
Chitosan with the deacetylation 90% of concentration 2g/100mL, the sodium β-glycerophosphate of concentration 50g/100mL by volume 1:1 mixes as blank matrix gel, again the slow-release microcapsule composition propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of this blank matrix gel with the propolis/hydroxypropylβ-cyclodextrin that contains 5g/100mL, 10g/100mL, three kinds of concentration of 20g/100mL.
Detect the one-tenth glue temperature of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber, gelation time and pH, result such as Fig. 6,7,8.Such as figure, the amount that the slow-release microcapsule of propolis/hydroxypropylβ-cyclodextrin adds blank matrix gel is larger, and the temperature of the one-tenth glue of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is higher, simultaneously, gelation time prolongs, 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, become the adjustment of glue temperature to return 37 ℃ it; When propolis/when hydroxypropylβ-cyclodextrin slow-release microcapsule concentration increased to 10g/100mL, it became the glue temperature just to be difficult to adjust back 37 ℃.Experiment is also found: in the situation that maintenance chitosan and sodium β-glycerophosphate consumption are constant, reduce the content of water in the thermosensitive hydrogel system, can effectively reduce one-tenth glue 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 such as table 14.
Table 14 water content is on the impact of gel-forming property
Drip the sodium hydroxide solution of 0.1mol/L to pH value about 7.13, investigate it and become glue temperature and gelation time, result such as table 15:
The single factor of table 15 is investigated the result
After in the chitosan thermosensitive hydrogel, adding as shown in Table 15 the propolis of recipe quantity 10g/100mL/hydroxypropylβ-cyclodextrin slow-release microcapsule, by reducing the content of water in the solution, the one-tenth glue temperature that raises can be recalled near 37 ℃ of the body temperatures, and can shorten its gelation time.
(2) preparation of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber
Requirement according to 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, the 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, the propolis of adding concentration 10g/100mL/hydroxypropylβ-cyclodextrin slow-release microcapsule, 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, places that 4 ℃ of environment are preserved, bubble to be eliminated is for subsequent use.
(3) investigation of the In Vitro Dissolution character of propolis flavone
By (2) identical method, the preparation 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 fully, preheating 10 min make it to form gel in 37 ± 0.5 ℃ of constant temperature oscillators, the artificial saliva 2mL that adds respectively again 37 ± 0.5 ℃ is release medium, frequency is 50r/min, amplitude is 20mm, respectively at after the vibration in 2,4,6,8,10,12,14,16,20, the 24h sampling.At the appointed time artificial saliva is all poured out, replenish immediately the 2mL artificial saliva.Sample filters with 0.45 μ m microporous filter membrane (water system), gets subsequent filtrate 1.5mL and lets cool, and with 2 times of artificial saliva dilution, shakes up.Measure the extracorporeal releasing quantity time dependent curve of absorbance in 5 different molecular weight propolis-chitosan periodontal slow release temperature sensing in situ gel rubber of propolis total flavones at the 415nm place with spectrophotography.The sample parallel assay of every curve three times is averaged, 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, discharges fully and approximately 20h is approaching 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 than 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.
The one-tenth assignment system of Fusayama Meyer type artificial saliva is adopted in above 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.Probe temperature: 37(± 0.5) ℃.
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 the propolis
Total flavones and total polyphenolsThe optimised process research [J] of extracting. the Academic Journal of Kunming Medical College, 2011,32 (12): 8-13.
Claims (5)
1. propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is characterized in that this gel:
(1) the chitosan acetic acid solution take concentration as 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) the hydroxypropylβ-cyclodextrin aqueous solution enclose propolis take concentration as 10g/100mL, the mass ratio between hydroxypropylβ-cyclodextrin aqueous solution and the 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 forms with step (2) propolis/slow-release microcapsule of hydroxypropylβ-cyclodextrin propolis by the temperature sensing in situ gel rubber of the blank substrate of step (1).
2. propolis-chitosan periodontal slow release temperature sensing in situ gel rubber according to claim 1, it is characterized in that: it is 90% that step (1) is further selected deacetylation, and molecular weight is 900,000 chitosan.
3. propolis-chitosan periodontal slow release temperature sensing in situ gel rubber according to claim 1 and 2, it is characterized in that: the one-tenth glue temperature of step (3) propolis-chitosan periodontal slow release temperature sensing in situ gel rubber is 36 ℃, gelation time is 65 seconds, the slow-release microcapsule stripping of propolis/hydroxypropylβ-cyclodextrin is slowly steady in this thermosensitive hydrogel, and propolis release in 37 ± 0.5 ℃, 24h reaches more than 99%.
4. such as the preparation method of one of the described propolis-chitosan periodontal of claim 1 ~ 3 slow release temperature sensing in situ gel rubber, it is characterized in that:
(1) the blank substrate temperature sensing in situ gel rubber of preparation: 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, and 4 ℃ leave standstill 1h; Get sodium β-glycerophosphate with concentration 125g/100mL and be dissolved in the distilled water, be stirred to dissolving, 4 ℃ leave standstill 1h; Sodium β-glycerophosphate solution is dropwise added chitosan solution, continue to stir 15min and fully mix, leave standstill to bubble collapse;
(2) slow-release microcapsule of preparation propolis/hydroxypropylβ-cyclodextrin propolis: put 95% ethanol 8mL in beaker, in 60 ℃ of water-bath 45min, be dissolved as propolis solution behind the adding 20g propolis; Get hydroxypropylβ-cyclodextrin adding dissolved in distilled water and get the cyclodextrin solution that concentration is 10g/100mL; Propolis solution is splashed into cyclodextrin solution, under temperature 50 C, stir 120 minutes to get mixture, again mixture is passed through 0.45 μ m microporous filter membrane filtering ethanol, obtain settled solution, leave standstill, lyophilization 4h, get 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 step (2) propolis/hydroxypropylβ-cyclodextrin slow-release microcapsule, when evenly being mixed to without obvious agglomerate, both survey pH, drip the 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.
5. the preparation method of propolis-chitosan periodontal slow release temperature sensing in situ gel rubber according to claim 4 is characterized in that: step (1) selects further that deacetylation is 90%, molecular weight is 900,000 chitosan.
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| WO2021015703A3 (en) * | 2019-07-24 | 2021-04-29 | Firat Universitesi Rektorlugu | Nanopropolis synthesis method with healing effect against side effects of cancer drug cisplatin |
| CN110664725A (en) * | 2019-11-22 | 2020-01-10 | 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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