CN106309380A - Icariin controlled release microsphere, preparation method thereof and application thereof - Google Patents
Icariin controlled release microsphere, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention discloses an icariin controlled release microsphere, a preparation method thereof and application thereof. The controlled release microsphere is used for pH response controlled release microsphere for treating colon disease, high-molecular polymers including chitosan, sodium alginate and like are used as carrier materials, the icariin is used as the active ingredient to construct an oral colon specific drug delivery system with biological controlled release targeting ability. The biological controlled release targeting microsphere disclosed by the invention has good pH responsiveness, the drug release amount under stomach low pH environment is low, the drug release can be promoted under the colon high pH environment, and the colon residence time of the drug is prolonged; the drug absorption is increased, and the local bioavailability of the oral drug for treating the colon disease is improved.
Description
Technical field
The invention belongs to pharmaceutical sanitary field, relate to the preparation method and applications of a kind of novel pharmaceutical formulation medicine microspheres,
The targeted controlled-release microspheres responded particularly to a kind of pH, its preparation method, medicinal application, this medicine microspheres is used for preventing, alleviating
And/or treatment knot intestinal disease, the disease such as including irritable bowel syndrome, ulcerative colitis, crohn, rectal cancer.
Background technology
Along with making constant progress of society, work, life stress increase, and work and rest diet is irregular, and long-term spirit is burnt
Consider, nervous etc., often lead to the health problem such as immune disorder and Flora Disturbance.The traditional Chinese medical science thinks that ulcerative colitis belongs to
Have loose bowels, the category such as spouting bleeding from anus, and modern medicine thinks that it belongs to autoimmune disease or with infection, with stomachache, diarrhoea, mucus
Just, bloody purulent stool etc. is clinical manifestation, changes with the chronic inflammatory disease of colonic mucosa and ulcer is as pathological characters.At present about bursting
The concrete cause of disease of ulcer colitis, pathogenesis is still not clear, and the most still lacks effective remedy measures.Mainly use glucocorticoid
It is treated by class, nonsteroidal anti-inflammatory drug or immunosuppressant, but poor effect.
Icariin is the principle active component of Berberidaceae plant Herba Epimedii, has defying age, antitumor, resistive connection enteritis etc.
Pharmacological action.But icariin poorly water-soluble, own biological availability is low, these factors limit its clinically should
With.In order to ensure the valid density of medicine, it is to avoid the toxic and side effects of medicine, reach effective therapeutic dose, the slow control of Chinese medicine modern times
Release formulation arises at the historic moment.Oral colon-specific drug release system (oral colon specific delivery system,
OCDDS) it is a kind of site-specific delivery of drugs system grown up the nineties in 20th century, i.e. refers to use suitable method, it is to avoid medicine exists
Stomach duodenum, jejunum and the release of ileum front end, be directly taken human body return, behind caecum position release and play local or complete
A kind of medicine-releasing system of body therapeutical effect, has the specificly-response to colon local environment, reasonable in design and preparation technology
The feature such as simple.
Chitosan is the deacetylated product of chitin, is unique natural alkaline polysaccharide, have widely biological activity and
Pharmaceutical researchers is enjoyed to pay close attention to.The positive charge of amino and saliva on alimentary canal mucous membrane on D-Glucose unit in chitosan molecule
Electrostatic attraction between the negative charge of acid residue makes it be easily adhered to gastrointestinal tract mucosa surface.And in chitosan molecule
Glucosides bond energy is degraded by colonic enzyme, therefore can be as the use of colon released drug material.Additionally alginate can realize sodium from
Exchange between son and calcium ion, thus the most swelling and alginate is in intracolic degraded.Can according to polysaccharide material
To improve targeting specific, meet the Treatment need of patient and crosslinking can be selected by the feature of colon physiology environment degradable
Agent carries out crosslinking curing to the microsphere of parcel chitosan.Amido on chitosan different molecular chain and aldehyde radical crosslink reaction,
The imido grpup and the schiff bases structure that generate enhance cross-linked chitosan stability in acid condition, can reduce chitosan
The hydrogen bond action of itself, reduces its bioadhesive.Glutaraldehyde cross-linking is capable of chitosan microball and is not detained at stomach, passes through
Gastrointestinal motility arrives colon, thus plays the effect for the treatment of colitis.
Although colitis pathogeny is the most unclear, but the process that inflammatory cytokine is formed in mediation colitis
Played in effect be mathematical.It is thin that the mediation colitis that wherein proinflammatory factor IL-1 β, TNF-α etc. are well recognized as is fallen ill
Intracellular cytokine, it addition, after inflammatory factor induction i-NOS expresses successfully, prostaglandin can promote that leukocyte goes to inflammation part also
Promote the generation of NO, thus cause the expression of inflammatory reaction and COX-II to make body itself produce inflammation and pain.
And have research to point out that the formation of inflammation and the expression of COX-II play vital effect in colon oncogenic process.
Conventional junction intestinal diseases administering mode mainly has oral and two kinds of approach of rectally, and oral administration is due to the spy of colon
Different physiological structure, medicine is difficult to arrive colon site, although rectally can make medicine discharge in local, but for colon
Upper end disease can not effectively play a role, and icariin is loaded into chitosan/calcium alginate and delays control-release microsphere system by the present invention
Standby oral colon-specific drug release system, for the treatment of peptic ulcer, has no that it is reported at present.
Summary of the invention
It is an object of the invention to exist the deficiency of technology for the medicine of existing treatment colonic diseases, it is provided that a kind of pH response
For the control-release microsphere treating colonic diseases and preparation method thereof, the control-release microsphere of the present invention has good pH response,
Can target administration, colon targeting controlled release microsphere prepared by the inventive method be mainly used in prevention, alleviate and/or treat colon class
Disease, including irritable bowel syndrome, ulcerative colitis, crohn, the purposes of the associated conditions such as rectal cancer.
For achieving the above object, the present invention provides the preparation method of a kind of control-release microsphere for treating colonic diseases, bag
Include the step that order below is carried out:
1) preparation emulsifying aqueous phase
In sodium alginate soln, add calcium carbonate, icariin, stirring, make calcium carbonate, icariin be suspended in Sargassum
In acid sodium solution, prepare calcium carbonate-icariin-sodium alginate suspension (i.e. emulsifying aqueous phase)；
2) preparation emulsifying oil phase
Emulsifying agent is joined in liquid paraffin, stirring, mixing, make emulsifying agent-paraffin solution (i.e. emulsifying oil phase)；
3) emulsifying
Under stirring, calcium carbonate-icariin-sodium alginate suspension is mixed with emulsifying agent-paraffin solution, enters
Row emulsifying, forms emulsification system；It is subsequently added into acid initiator, carries out acid initiation and process, generate icariin-calcium alginate
Microsphere；
4) icariin-calcium alginate microsphere is joined in film forming chitosan solution, mix homogeneously, carry out multiple electrolyte
Complex reaction, chitosan is wrapped in the surface of icariin-calcium alginate microsphere, makes chitosan-icariin-alginic acid
Calcium microsphere；
5) chitosan-icariin-calcium alginate microsphere is mixed with cross-linking agent, carry out cross-linking reaction, obtain Herba Epimedii
Glycosides control-release microsphere.
Wherein, step 1) described in the weight of calcium carbonate be 0.2-2:100 with the ratio of the volume of sodium alginate soln, preferably
For 0.5-1.5:100, more preferably 1:100, i.e. every 100ml sodium alginate soln adds 0.2-2g nanometer grade calcium carbonate
Or every 1L sodium alginate soln adds 2-20g calcium carbonate；The weight of described icariin and the volume of sodium alginate soln it
Ratio is 1-3:100, preferably 1.5-2.5:100, more preferably 2.25:100, adds in i.e. every 100ml sodium alginate soln
Enter in 1-3g icariin or every 1L sodium alginate soln and add 10-30g icariin.
Particularly, described calcium carbonate selects nanometer grade calcium carbonate.
Wherein, the concentration of described sodium alginate soln is 0.5-2% (w/v), preferably 1-2%, more preferably
1.5%.
Particularly, described sodium alginate soln is prepared from accordance with the following steps: joined by sodium alginate in pure water, mixed
Close, under the conditions of being heated and maintained at 45 ± 5 DEG C, stirring, make sodium alginate fully dissolve, to obtain final product, wherein, in sodium alginate soln
The quality of sodium alginate is 0.5%-2% (w/v) with the concentration that ratio is 0.5-2:100, i.e. sodium alginate soln of the volume of water.
Wherein, step 2) described in emulsifying agent select sorbester p17 or/and Tween 80.
Particularly, described emulsifying agent is 0.5-2:100, preferably 0.5-1.5:100 with the ratio of the volume of liquid paraffin, enters
One step is preferably 1.0:100.
Wherein, step 3) described in emulsifying temperature be 35-45 DEG C, preferably 37 DEG C；The emulsifying time is 30-
60min, preferably 40-50min.
Particularly, the ratio of described calcium carbonate-icariin-sodium alginate suspension and the volume of emulsifying agent-paraffin solution
For 1:4-6, preferably 1:5.
Especially, described stir speed (S.S.) is 200-500rpm, preferably 300rpm.
Wherein, described acid initiator selects glacial acetic acid, hydrochloric acid, sulphuric acid, phosphoric acid, preferably glacial acetic acid.
Particularly, described acid initiator is the mixed solution of glacial acetic acid and liquid paraffin, wherein glacial acetic acid and acid initiator
The ratio of volume be 1:8-12, preferably 1:10.
Especially, the volume of glacial acetic acid and carbon in calcium carbonate-icariin-sodium alginate suspension in described acid initiator
The weight ratio of acid calcium is 5:1-3, preferably 5:1, i.e. the every carbon Han 1-3g in calcium carbonate-icariin-sodium alginate suspension
Glacial acetic acid 5ml in the sour initiator that acid calcium then adds, or often containing 1-in calcium carbonate-icariin-sodium alginate suspension
Glacial acetic acid 5L in the sour initiator that 3kg calcium carbonate then adds.
Wherein, described acid initiation treatment temperature 35-45 DEG C, preferably 37 DEG C；The acid initiation process time is 10-20min, excellent
Elect 15min as.
Particularly, also include that the mixed system after processing acid initiation carries out standing process, lower leaf on mixed system, take
Lower sediment, obtains described icariin-calcium alginate microsphere.
Especially, employing volume by volume concentration is the Tween80 aqueous cleaning precipitation of 0.5-2% (v/v), unnecessary to remove
Liquid paraffin；The most again with pure water, collect icariin-calcium alginate microsphere.
Wherein, step 4) described in film forming chitosan solution be chitosan-acetum.
Particularly, in described chitosan-acetum, the concentration of chitosan is 0.5%-2% (w/v), preferably 1%.
Especially, described film forming chitosan solution is the most formulated: add chitosan into glacial acetic acid water
In solution, after mixing, heat temperature raising under the conditions of being maintained at 45 ± 5 DEG C, stirring, make chitosan fully dissolve, after it cools down
The pH of regulation chitosan-acetum is 5-6, standby.
Wherein, in described chitosan-acetum, the quality of chitosan is 0.5-with the ratio of the volume of glacial acetic acid aqueous solution
In 2:100, i.e. chitosan-acetum, the concentration of chitosan is 0.5%-2% (w/v), preferably 1:100.
Particularly, described glacial acetic acid aqueous solution concentration of volume percent is 0.5-2% (v/v), preferably 1%.
Especially, the pH of regulation chitosan-acetum is 5.5.
Wherein, the time >=20min, preferably 20-40min of described multiple electrolyte complex reaction.
Particularly, icariin-calcium alginate microsphere is 1:5-15 with the ratio of the volume of film chitosan solution, preferably 1:
8。
Especially, the reaction system after also including multiple electrolyte complex reaction is centrifuged processing, then to centrifugal heavy
Shallow lake thing carries out water cleaning.
Wherein, step 5) described in cross-linking agent select the one in glutaraldehyde water solution, PBS, preferably penta 2
Aldehyde aqueous solution.
Particularly, the mass percent concentration of described glutaraldehyde water solution is 0.25-1%, preferably 0.5%.
Especially, described chitosan-icariin-calcium alginate microsphere is 1:5-20 with the ratio of the volume of cross-linking agent, excellent
Elect 1:10 as.
Wherein, and the temperature of described cross-linking reaction < 10 DEG C, preferably 4-10 DEG C, more preferably 4-8 DEG C, further
It is preferably 4 DEG C；Cross-linking reaction time is 1.5-3h, preferably 2h.
Particularly, also include adding crosslinking terminator, the cross-linking reaction described in termination.
Wherein, described crosslinking terminator selects glycine solution.
Particularly, the molar concentration of described glycine solution is 0.5-2mol/L, preferably 1mol/L.
Especially, the glycine solution of described addition is 2-4:1, preferably 2:1 with the ratio of the volume of glutaraldehyde solution.
Particularly, also include that the mixed system after terminating cross-linking reaction is centrifuged processing, then to centrifugal sediment
It is carried out with pure water, is then dried, to obtain final product.
Another aspect of the present invention provides a kind of icariin control-release microsphere being prepared from according to the method described above.
Further aspect of the present invention provides a kind of icariin control-release microsphere being prepared from according to the method described above to control in preparation
Treat the application in colonic diseases, the application in the medicine or health product of preparation treatment colonic diseases.
The biological controlled-released microsphere of icariin prepared by the present invention has the advantage that
1, the biological controlled-released microsphere of the icariin of the present invention is that pH responds control-release microsphere, has good pH response,
Not occurring under stomach low ph conditions significantly to degrade, release amount of medicine is relatively low, sticks to colon table under colon higher pH environment
Face sustained release drugs, it is possible to promotion drug release, and extend the colon holdup time of medicine, increase drug absorption, improve mouth
Take the local biologic availability of medicine treatment colon site disease.Tablets in vitro result of study shows, the slow control of icariin targeting
Release accumulative release rate only about 10% after discharging 2h in microsphere simulated gastric fluid, and simulate accumulative release rate in colonic fluid and can reach
65%.
The core of the biological controlled-released microsphere of icariin that 2, prepared by the inventive method is icariin calcium alginate microsphere, outward
Shell is chitosan, has preferable pH response controlled-release effect and icariin colon targeting controlled release drug action simultaneously, is used for controlling
Treat colonic diseases, treat the diseases such as irritable bowel syndrome, ulcerative colitis, crohn, rectal cancer.
Icariin targeting sustained and controlled release microsphere prepared by the inventive method can significantly improve TNBS/ ethanol-induced ulcerative
Colitis, it is possible to effectively reduce the secretion of colon site inflammatory factor IL-1 β, IL-6, reduce cause inflammatory reaction i-NOS,
COX-II protein expression level.
3, the icariin pH of the present invention responds biological controlled-released microsphere drug and is carried on microsphere internal core, drug delivery amount
Height, the outer layer covers chitosan of medicine, it is possible to fixed drug, thus reduce the loss in medicine transportation in vivo.
4, the icariin pH of the present invention responds the target administration effect of biological controlled-released microsphere substantially, and microsphere is at colon site
Long-time stop, improves therapeutic effect.In vivo study shows that microsphere can stop more than 12h at colon, is more beneficial for medicine
Release and performance therapeutical effect.
5, for chitosan outside the icariin targeting sustained and controlled release microsphere that prepared by the present invention, for pH sensitive cationic type water
Gel rubber material, has good bioadhesive, biocompatibility and biodegradability, to body avirulence, and has anti-
Bacterium antiinflammatory, promotion wound healing effect etc..
6, the inventive method easy control of process conditions, the product quality of preparation is controlled, big production suitable for industrialized.
Accompanying drawing explanation
Fig. 1 icariin chitosan/calcium alginate control-release microsphere is the accumulative release rate of (pH=1.2) in simulated gastric fluid
Figure.
Fig. 2 icariin chitosan/calcium alginate control-release microsphere is at simulated intestinal fluid and colonic fluid (pH=6.8, pH=
7.4) the preparation figure in.
Fig. 2 A is icariin/chitosan/calcium alginate microsphere preparation figure in simulated intestinal fluid (pH=6.8).
Distribution results figure in Fig. 3 FITC fluorescent-labeled microspheres mice digestive tract, is stomach, 12 fingers the most respectively
Intestinal, jejunum and colon.
Fig. 4 icariin chitosan/calcium alginate control-release microsphere is to Ulcerative Colitis Model rat colon tissue IL-1 β
Content affect result figure.
Fig. 5 icariin chitosan/calcium alginate control-release microsphere is to Ulcerative Colitis Model rat colon tissue T NF-α
Content affect result figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.But these embodiments be only limitted to illustrate the present invention and not
For limiting the scope of the present invention.The experimental technique of unreceipted specific experiment condition in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.
Of the present invention icariin targeting sustained and controlled release microsphere is expanded on further for colitis below by way of test example
Therapeutical effect, these test examples include the preparation of icariin targeting sustained and controlled release microsphere of the present invention and drug release test and
The pharmacodynamics test of icariin targeting sustained and controlled release microsphere of the present invention.
The reagent used in the embodiment of the present invention is as follows:
The preparation of embodiment 1 Herba Epimedii/glycosides chitosan/calcium alginate microsphere
1, preparation solution
1-1) preparation sodium alginate soln
The sodium alginate of accurate weighing is joined in pure water, mixing, under the conditions of being heated and maintained at 45 ± 5 DEG C, stirring,
Make sodium alginate fully dissolve, prepare sodium alginate soln, in 4 DEG C of Refrigerator stores after it is down to room temperature (about 20-25 DEG C),
Being heated to 45 DEG C before using, standby, wherein, in sodium alginate soln, the quality of sodium alginate is 0.5 with the ratio of the volume of water:
100, i.e. the concentration of sodium alginate soln is 1.0% (w/v)；
1-2) it is configured to coating solution (i.e. chitosan-acetum)
The chitosan of accurate weighing is joined in glacial acetic acid (v/v, the concentration of volume percent) aqueous solution of 0.5%, mixed
After even, heat temperature raising under the conditions of being maintained at 45 ± 5 DEG C, stirring, make chitosan fully dissolve, prepare chitosan-acetum
(i.e. film forming solution), in 4 DEG C of Refrigerator stores after it is down to room temperature (about 20-25 DEG C), before using, regulation chitosan-acetic acid is molten
The pH of liquid is 5.5, standby, and wherein, in chitosan-acetum, the quality of chitosan with the ratio of the volume of glacial acetic acid aqueous solution is
In 0.5:100, i.e. chitosan-acetum, the concentration of chitosan is 0.5% (w/v)；
The concentration of volume percent of glacial acetic acid aqueous solution is in addition to 0.5%, and other concentration 0.5-2.0% are all applicable to this
Invention；The pH value of the chitosan-acetum before present invention use illustrates as a example by 5.5, and other pH value 5-6 is all applicable to
The present invention.
1-3) preparating acid initiator
The glacial acetic acid of accurate measuring is joined in liquid paraffin, stirring, mix homogeneously, standby, wherein glacial acetic acid and acid
The ratio of the volume of initiator is 1:8-12, preferably 1:10.
In the embodiment of the present invention, in acid initiator, the ratio of glacial acetic acid and the volume of liquid paraffin illustrates as a example by 1:10,
Other glacial acetic acids are that 1:8-12 is all applicable to the present invention with the ratio of the volume of liquid paraffin.
2, icariin-calcium alginate microsphere is prepared
2-1) preparation aqueous phase
With syringe removing step 1) sodium alginate soln (20ml) prepared is placed in beaker, electromagnetic agitation, protects in temperature
Holding and add nanometer grade calcium carbonate (0.1g) under the conditions of being 45 ± 5 DEG C, be stirring evenly and then adding into icariin (0.3g), stirring is all
Even so that nanometer grade calcium carbonate and icariin are suspended in sodium alginate soln, prepare calcium carbonate-icariin-alginic acid
Sodium solution (i.e. aqueous phase, 20ml)；Wherein: the weight of calcium carbonate is 0.5:100 with the ratio of the volume of sodium alginate soln；Herba Epimedii
The weight of glycosides is 1.5:100 with the ratio of the volume of sodium alginate soln.
2-2) preparation oil phase
Pipette surfactant Span 80 (0.5ml) with syringe and join in liquid paraffin (100ml), in 37 ± 5 DEG C
Under water bath condition, stirring so that it is fully mix, prepare Span-paraffin solution (oil phase) standby, wherein surfactant Span
80 is 0.5:100 with the ratio of the volume of liquid paraffin；
Illustrating as a example by surfactant Span80 in the embodiment of the present invention, other surfactants are all applicable to this
Invention, such as Tween80 etc..
2-3) acid initiation processes
Under conditions of 37 ± 1 DEG C of water-baths, mixing speed are 300rpm (200-500rpm is also suitable), aqueous phase is joined
Carrying out emulsifying in oil phase, stirring makes aqueous phase fully mix with oil phase, forms emulsification system；
(5mL contains to add acid initiator liquid paraffin-acetic acid mixed liquor after emulsifying 45min immediately in emulsification system
Glacial acetic acid 0.5ml), carry out acid initiation process, formed icariin-calcium alginate microsphere, wherein, acid initiator in glacial acetic acid with
The ratio of the volume of liquid paraffin is 1:10；In acid initiator, the volume of glacial acetic acid is 5:1 with the weight ratio of calcium carbonate in aqueous phase
(v/w)；
Acid initiation stops stirring after processing 15min, and the most static placement, lower leaf on mixed system (i.e. stratification) is quiet
After putting 30min, take off a layer icariin-calcium alginate microsphere precipitation, with the Tween80 aqueous cleaning 3 times of volume ratio 0.5%
Precipitation, to remove unnecessary liquid paraffin, then with pure water, until cleaning up, collects icariin-calcium alginate micro-
Ball.
In the embodiment of the present invention, aqueous phase illustrates as a example by 1:5 with the ratio of the volume of oil phase, other aqueous phases and oil phase
The ratio of volume is that 1:4-6 is all applicable to the present invention；In acid initiator, glacial acetic acid and the ratio of the volume of liquid paraffin are as a example by 1:10
Illustrating, other proportionings such as 1:9-11 is all applicable to the present invention
The present invention using glacial acetic acid with the mixed liquor being mixed with liquid paraffin as acid initiator, can be by photoinitiate acid
Mixing homogeneously with emulsification system, glacial acetic acid is rapidly and evenly distributed in emulsification system, it is to avoid locally acid concentration is too high or too low,
The glacial acetic acid being simultaneously introduced and nano-calcium carbonate generation chemical reaction, make calcium in nano-calcium carbonate dissociate with the state of ion
Coming, the sour initiator liquid paraffin-acetic acid mixed liquor of addition forms a buffer solution system in emulsification system, makes calcium ion
Release that can be slow, lasting, the calcium ion of release reacts with the sodium alginate in emulsification system microemulsion, forms internal friendship
Connection, generates calcium alginate and is uniformly distributed in the microsphere of formation, thus it is micro-to advantageously form uniform particle sizes, hard-packed micron order
Ball.
During emulsifying of the present invention forms icariin-calcium alginate microsphere, glacial acetic acid is used to cause nano-scale carbon
Acid calcium release calcium ion, not only makes preparation process condition of the present invention reaction gentleness, and controllability is strong, advantageously forms uniform particle sizes
Micron order microsphere；And owing to being uniformly distributed in inside microemulsion due to nano-calcium carbonate in emulsion process, cause acetic acid
Release calcium ion and alginic acid molecule form gel, and the gel so formed belongs to internal crosslinking so that the gel micro-ball of formation
Structure is the tightst, has good medicine controlled releasing performance；And other are prepared microsphere and are typically employed in after microemulsion formed, then add
Add calcium chloride or other ionic calcium soln solidify to form gel, due to microemulsion aqueous phase droplets diameter at 100 microns even more
Greatly, touch calcium ion outside microemulsion at first, solidify rapidly, define a dense gel " shell ", be unfavorable for
Calcium ion internally migrates, and affects curing efficiency so that fail inside the microsphere of formation to solidify very well, but a microcapsule,
Drug release so can be caused too fast or the prominent release thing of microcapsule disintegration, be unfavorable for colon targeting drug administration.
3, the preparation of chitosan-icariin-calcium alginate microsphere
By accurate measuring step 2) icariin-calcium alginate microsphere of preparing joins film forming solution chitosan-acetic acid
In solution (pH5.5), making chitosan and sodium alginate that multiple electrolyte complex reaction occurs under stirring, chitosan is wrapped in
The surface of icariin-calcium alginate microsphere, after magnetic agitation 40min, makes chitosan fully and sodium alginate occurs telegram in reply to solve
Matter complex reaction, is wrapped in the surface of icariin calcium alginate microsphere.Centrifugal collection microsphere, cleans with pure water after completion of the reaction
Microsphere surface, removes unreacted chitosan solution, is collected by microsphere, it is thus achieved that the chitosan-icariin-sea after complex reaction
Calcium alginate microsphere, wherein, icariin-calcium alginate microsphere is 1:5 with the volume ratio of film forming solution；Described multiple electrolyte complexation
Response time is 40min.
4, the preparation of icariin/chitosan/calcium alginate control-release microsphere
(mass percent concentration is 4-1) chitosan-icariin-calcium alginate microsphere to be joined cross-linking agent solution
The glutaraldehyde water solution of 0.25%) in, stirring, mix homogeneously, under the conditions of 4 DEG C, shake on roller bearing, glutaraldehyde gathers with shell
Sugar-icariin-calcium alginate microsphere crosslinks reaction, wherein, and chitosan-icariin-calcium alginate microsphere and penta 2
The ratio of the volume of aldehyde solution is 1:5；
In the embodiment of the present invention, cross-linking reaction temperature illustrates as a example by 4 DEG C, it can be avoided that glutaraldehyde under cryogenic conditions
With chitosan generation vigorous reaction so that more slowly steadily, the microsphere surface thickness of formation is more uniform for cross-linking reaction speed
Unanimously, other temperature are all applicable to the present invention less than under the conditions of 10 DEG C (such as 4-8 DEG C).
After 4-2) cross-linking reaction time is 1.5h, adding crosslinking terminator molar concentration in reaction system is 0.5mol/L
Glycine solution remove unreacted glutaraldehyde, wherein, the consumption of glycine solution and glutaraldehyde solution volume ratio are 2:1；
4-3) above-mentioned solution is obtained by centrifuge the icariin control-release microsphere of hygrometric state, then cleans with pure water
Microsphere surface, recentrifuge, 3 times repeatedly, finally by standby for the icariin control-release microsphere lyophilization 24h that obtains.
Embodiment 2
1, preparation solution
1-1) preparation sodium alginate soln
The sodium alginate of accurate weighing is joined in pure water, mixing, under the conditions of being heated and maintained at 45 ± 5 DEG C, stirring
Make sodium alginate fully dissolve, prepare sodium alginate soln, in 4 DEG C of Refrigerator stores after it is down to room temperature, be heated to before using
45 DEG C, standby, wherein, in sodium alginate soln, the quality of sodium alginate is 1.5% with the ratio of the volume of water；
1-2) preparation chitosan-acetum (film forming solution)
The chitosan of accurate weighing is joined in glacial acetic acid (v/v, the concentration of volume percent) aqueous solution of 1%, mixing
After, heat temperature raising under the conditions of being maintained at 45 ± 5 DEG C, stirring, make chitosan fully dissolve, prepare chitosan-acetum
(film forming solution), in 4 DEG C of Refrigerator stores after it is down to room temperature, before using, the pH of regulation chitosan-acetum is 5.5, standby
With, wherein, in chitosan-acetum, the quality of chitosan and the ratio of the volume of glacial acetic acid aqueous solution are that 1:100, i.e. shell are poly-
In sugar-acetum, the concentration of chitosan is 1%；
The concentration of volume percent of glacial acetic acid aqueous solution is in addition to 1%, and other concentration 0.5-2.0% are all applicable to this
Bright；The pH value of the chitosan-acetum before present invention use is in addition to 5.5, and other pH value 5-6 is all applicable to the present invention.
1-3) preparating acid initiator
The glacial acetic acid of accurate measuring is joined in liquid paraffin, stirring, mix homogeneously, standby, wherein glacial acetic acid and acid
The ratio of the volume of initiator is 1:10.
2, icariin-calcium alginate microsphere is prepared
2-1) preparation aqueous phase
With syringe removing step 1) sodium alginate soln (20ml) prepared is placed in beaker, electromagnetic agitation, protects in temperature
Holding and add nanometer grade calcium carbonate (0.2g) under the conditions of being 45 ± 5 DEG C, be stirring evenly and then adding into icariin (0.45g), stirring is all
Even so that nanometer grade calcium carbonate and icariin are suspended in sodium alginate soln solution, prepare calcium carbonate-icariin-sea
Solution of sodium alginate (i.e. aqueous phase, 20ml)；Wherein: the weight of calcium carbonate is 1:100 with the ratio of the volume of sodium alginate soln；Excessive sheep
The weight of icariin is 2.25:100 with the ratio of the volume of sodium alginate soln.
2-2) preparation oil phase
Pipette surfactant Span 80 (1ml) with syringe and join in liquid paraffin (100ml), in 37 ± 5 DEG C of water
Under the conditions of bath, mechanical agitation so that it is fully mix, prepare Span-paraffin solution (oil phase) standby, wherein surfactant
Span80 is 1:100 with the ratio of the volume of liquid paraffin；
2-3) acid initiation processes
Under conditions of 37 ± 1 DEG C of water-baths, mixing speed are 300rpm, aqueous phase are joined in oil phase and carry out at emulsifying
Reason, stirring makes aqueous phase fully mix with oil phase, forms emulsification system；
(5mL contains to add acid initiator liquid paraffin-acetic acid mixed liquor after emulsifying 40min immediately in emulsification system
Glacial acetic acid 0.5ml), carry out acid initiation and process, form icariin-calcium alginate microsphere, wherein, glacial acetic acid in acid initiator
Volume is 5:2 (v/w) with the weight ratio of calcium carbonate in aqueous phase；
Acid initiation stops stirring after processing 15min, and the most static placement, lower leaf on mixed system (i.e. stratification) is quiet
After putting 30min, take off a layer icariin-calcium alginate microsphere precipitation, sink for 3 times with the Tween80 aqueous cleaning of volume ratio 1%
Forming sediment, to remove unnecessary liquid paraffin, then with pure water, until cleaning up, collecting icariin-calcium alginate micro-
Ball.3, the preparation of chitosan-icariin-calcium alginate microsphere
By accurate measuring step 2) icariin-calcium alginate microsphere of preparing joins film forming solution chitosan-acetic acid
In solution (pH5.5), making chitosan and sodium alginate that multiple electrolyte complex reaction occurs under stirring, chitosan is wrapped in
The surface of icariin-calcium alginate microsphere, after magnetic agitation 30min, makes chitosan fully and sodium alginate occurs telegram in reply to solve
Matter complex reaction, is wrapped in the surface of icariin calcium alginate microsphere.Centrifugal collection microsphere, cleans with pure water after completion of the reaction
Microsphere surface, removes unreacted chitosan solution, is collected by microsphere, it is thus achieved that the chitosan-icariin-sea after complex reaction
Calcium alginate microsphere, wherein, icariin-calcium alginate microsphere is 1:8 with the volume ratio of film forming solution；Described multiple electrolyte complexation
Response time is 30min；
4, icariin/chitosan/calcium alginate controls the preparation of microsphere
(mass percent concentration is 4-1) chitosan-icariin-calcium alginate microsphere to be joined cross-linking agent solution
The glutaraldehyde solution of 0.5%) in, stirring, mix homogeneously, under the conditions of 4 DEG C, shake on roller bearing, glutaraldehyde gathers with parcel shell
Icariin-the calcium alginate microsphere of sugar crosslinks reaction, wherein, and chitosan-icariin-calcium alginate microsphere and penta 2
The ratio of the volume of aldehyde solution is 1:10；
After 4-2) cross-linking reaction time is 2h, in reaction system, adds glycine solution (molar concentration is 1mol/L) go
Except unreacted glutaraldehyde, wherein, the consumption of glycine solution and glutaraldehyde solution volume ratio are 2:1；
4-3) above-mentioned solution is obtained by centrifuge the icariin control-release microsphere of hygrometric state, then cleans with pure water
Microsphere surface, recentrifuge, 3 times repeatedly, finally by standby for the icariin control-release microsphere lyophilization 24h that obtains.
Embodiment 3
1, preparation solution
1-1) preparation sodium alginate soln
The sodium alginate of accurate weighing is joined in pure water, mixing, under the conditions of being heated and maintained at 45 ± 5 DEG C, stirring,
Make sodium alginate fully dissolve, prepare sodium alginate soln, in 4 DEG C of Refrigerator stores after it is down to room temperature, be heated to before using
45 DEG C, standby, wherein, in sodium alginate soln, the quality of sodium alginate is 2% with the ratio of the volume of water；
1-2) preparation chitosan-acetum (film forming solution)
The chitosan of accurate weighing is joined in glacial acetic acid (v/v, the concentration of volume percent) aqueous solution of 2%, mixing
After, heat temperature raising under the conditions of being maintained at 45 ± 5 DEG C, stirring, make chitosan fully dissolve, prepare chitosan-acetum (i.e.
Film forming solution), in 4 DEG C of Refrigerator stores after it is down to room temperature, before using, the pH of regulation chitosan-acetum is 5.5, standby
With, wherein, in chitosan-acetum, the quality of chitosan is 2% with the ratio of the volume of glacial acetic acid aqueous solution；
The concentration of volume percent of glacial acetic acid aqueous solution is in addition to 2%, and other concentration 0.5-2.0% are all applicable to this
Bright；The pH value of the chitosan-acetum before present invention use is in addition to 5.5, and other pH value 5-6 is all applicable to the present invention.
2, icariin-calcium alginate microsphere is prepared
2-1) preparation aqueous phase
With syringe removing step 1) sodium alginate soln (20ml) prepared is placed in beaker, electromagnetic agitation, protects in temperature
Holding and add nanometer grade calcium carbonate (0.3g) under the conditions of being 45 ± 5 DEG C, be stirring evenly and then adding into icariin (0.5g), stirring is all
Even so that nanometer grade calcium carbonate and icariin are suspended in sodium alginate soln solution, prepare calcium carbonate-icariin-sea
Solution of sodium alginate (i.e. aqueous phase, 20ml)；Wherein: the weight of calcium carbonate is 1.5:100 with the ratio of the volume of sodium alginate soln；Excessive
The weight of sheep icariin is 2.5:100 with the ratio of the volume of sodium alginate soln.
2-2) preparation oil phase
Pipette surfactant Span 80 (1.5ml) for syringe and join in liquid paraffin (100ml), in 37 ± 5
Under DEG C water bath condition, stirring so that it is fully mix, prepare Span-paraffin solution (oil phase) standby, wherein surfactant
Span80 is 1.5:100 with the ratio of the volume of liquid paraffin；
2-3) acid initiation processes
Under conditions of 37 ± 1 DEG C of water-baths, mixing speed are 300rpm, aqueous phase are joined in oil phase and carry out at emulsifying
Reason, stirring makes aqueous phase fully mix with oil phase, forms emulsification system；
(5mL contains to add acid initiator liquid paraffin-acetic acid mixed liquor after emulsifying 50min immediately in emulsification system
Glacial acetic acid 0.5ml), carry out acid initiation and process, form icariin-calcium alginate microsphere, wherein, liquid paraffin in acid initiator
It is 10:1 with the ratio of the volume of glacial acetic acid；In acid initiator, the volume of glacial acetic acid is 5:3 with the weight ratio of calcium carbonate in aqueous phase
(v/w)；
Acid initiation stops stirring after processing 20min, and the most static placement, lower leaf on mixed system (i.e. stratification) is quiet
After putting 30min, take off a layer icariin-calcium alginate microsphere precipitation, with the Tween80 aqueous cleaning 3 times of volume ratio 1.5%
Precipitation, to remove unnecessary liquid paraffin, then with pure water, until cleaning up, collects icariin-calcium alginate micro-
Ball.
3, the preparation of chitosan-icariin-calcium alginate microsphere
By accurate measuring step 2) icariin-calcium alginate microsphere of preparing joins film forming solution chitosan-acetic acid
In solution (pH5.5), making chitosan and sodium alginate that multiple electrolyte complex reaction occurs under stirring, chitosan is wrapped in
The surface of icariin-calcium alginate microsphere, after magnetic agitation 20min, makes chitosan fully and sodium alginate occurs telegram in reply to solve
Matter complex reaction, is wrapped in the surface of icariin calcium alginate microsphere.Centrifugal collection microsphere, cleans with pure water after completion of the reaction
Microsphere surface, removes unreacted chitosan solution, is collected by microsphere, it is thus achieved that the chitosan-icariin-sea after complex reaction
Calcium alginate microsphere, wherein, icariin-calcium alginate microsphere is 1:15 with the volume ratio of film forming solution；Described multiple electrolyte network
The conjunction response time is 20min.
4, icariin/chitosan/calcium alginate controls the preparation of microsphere
(mass percent concentration is 4-1) chitosan-icariin-calcium alginate microsphere to be joined cross-linking agent solution
The glutaraldehyde water solution of 1%) in, stirring, mix homogeneously, under the conditions of 8 DEG C, shake on roller bearing, glutaraldehyde and chitosan-excessive
Sheep icariin-calcium alginate microsphere crosslinks reaction, wherein, and chitosan-icariin-calcium alginate microsphere and glutaraldehyde solution
The ratio of volume be 1:20；
After 4-2) cross-linking reaction time is 3h, in reaction system, adds glycine solution (molar concentration is 2mol/L) go
Except unreacted glutaraldehyde, wherein, the consumption of glycine solution and film forming solution volume ratio are 1:1；
4-3) above-mentioned solution is obtained by centrifuge the icariin control-release microsphere of hygrometric state, then cleans with pure water
Microsphere surface, recentrifuge, 3 times repeatedly, finally by standby for the icariin control-release microsphere lyophilization 24h that obtains.
Embodiment 4 icariin/chitosan/calcium alginate control-release microsphere external release drug study
According to 2015 editions " Chinese Pharmacopoeias " annex, the first method " Rotating shaker " that " release " measures measures, point
Not with simulated gastric fluid (2h, pH=1.2)；Simulated intestinal fluid (3h, pH=6.8)；Simulation colonic fluid (24h, pH=7.4) is dissolution
Medium, temperature is 37 ± 0.5 DEG C, and rotating speed is 50r/min.
The accurate icariin weighing embodiment 2 preparation delays each 2g of control-release microsphere respectively, totally 8 parts, puts into 8 dry turning
In basket, simulated gastric fluid 4 parts；Simulated intestinal fluid, simulation colonic fluid 4 parts, regulation turns basket rotating speed 50rpm, after it is steady, will turn basket
Fall in the stripping rotor containing 200mL dissolution medium (simulated gastric fluid, simulated intestinal fluid) of constant temperature, contact dissolution medium from sample
Playing timing immediately, in the separately sampled 1mL of different time points, wherein simulated gastric fluid sampling time point is 5,10,15,20,25,30,
40,50,60,80,100,120min；Simulated intestinal fluid sampling time point is 5,10,15,20,25,30,40,50,60,80,
100,120,150,180min；The icariin prepared in the present invention delays after control-release microsphere discharges medicine 3h in simulated intestinal fluid
The basket that turns that will be equipped with icariin control-release microsphere is transferred to simulate colonic fluid from simulated intestinal fluid, and carry out prepared by the present invention is excessive
Sheep icariin delays control-release microsphere drug release test in simulation colonic fluid, and the sampling time point of simulation colonic fluid is: 200,
260,320,400,600,720,1440,2880min, add the simulated solution of same volume after every sub-sampling simultaneously, sample will be obtained
Product use high-efficient liquid phase color law popularization (HPLC) to measure the content of icariin in release liquid after 0.45 μm filtering with microporous membrane.
Icariin is delayed control-release microsphere in simulated intestinal fluid, first discharges 180min, be then placed in simulating in colonic fluid
Continuing release, mainly see its release conditions under microsphere arrives colonic environment, due to the special construction of colon, microsphere is wherein
Residence time is longer, also extends release time, extend to 2800min when that therefore we simulating.
Icariin/chitosan/calcium alginate microsphere after discharging 2h in simulated gastric fluid accumulative release rate only have 10% (as
Fig. 1), Fig. 2 shows microsphere release conditions in simulation small intestinal and colonic fluid, and result shows icariin control prepared by the present invention
Releasing microsphere can discharge in a large number at simulation small intestinal and colonic fluid Chinese medicine, accumulative release rate can reach 65%, in higher ph
In simulation colonic fluid, icariin/chitosan/calcium alginate control-release microsphere can be with sustained release drugs.
In Fig. 2, the dotted line left side is the drug release situation of simulated intestinal fluid (pH 6.8), and drug release time is from 5-180min；Empty
The right of line is expressed as simulating the drug release situation of colonic fluid (pH7.4), and drug release time is from 200-2880min.
Fig. 2 A is icariin/chitosan/calcium alginate microsphere release conditions in simulated intestinal fluid, prepared by the present invention
Icariin control-release microsphere accumulative release rate after release 180min is 10.51%.
It is distributed in embodiment 5 Herba Epimedii/glycosides chitosan/digested road of calcium alginate controlled release microspheres
Take the icariin-calcium alginate microsphere according to the preparation of embodiment 2 step 2 of certain volume, by icariin-sea
Calcium alginate microsphere joins in the FITC fluorescently-labeled film forming solution chitosan-acetum (pH5.5) of 6 times, magnetic agitation
30min, after completion of the reaction, with 70% methanol solution wash microsphere to supernatant 495nm without absorb, then with pure water clean micro-
Ball surface, obtains FITC labelling chitosan-icariin-calcium alginate microsphere；Wherein, the fluorescently-labeled film forming of described FITC is molten
In liquid, the concentration of the fluorescently-labeled chitosan of FITC is 10mg/mL；
FITC labelling chitosan-icariin-calcium alginate microsphere is scattered in the 0.5% glutaraldehyde cross-linking agent of 10 times
In, it is placed on roller bearing shaking cross-linking reaction 2h under the conditions of 4 DEG C.Unnecessary unreacted penta 2 are washed away with the glycine solution of 1mol/L
Aldehyde solution, finally cleans microsphere surface with pure water, lyophilization 24h, prepare the fluorescently-labeled icariin/chitosan of FITC/
Calcium alginate control-release microsphere, standby.
The male ICR mouse choosing health is tested, body weight 20 ± 2g, fasting 24h before experiment, freely drinks water；Will
Icariin/chitosan/calcium alginate the control-release microsphere of the FITC labelling arrived, is uniformly dispersed with distilled water, according to 6mg/20g
(weight/mice weights of microsphere) mouse stomach gives the icariin/chitosan/calcium alginate control-release microsphere of FITC labelling,
Totally 7 groups, often 3 mices of group, respectively different time points after gavage (2,4,6,12,16,24,36h), by experimental mice fiber crops
Separating Stomach duodenum, jejunum, colon four part after liquor-saturated, 2h is first group, and 4h is second group, and 6h is the 3rd group, and 12h is
Four groups, 16h is the 5th group, and 24h is the 6th group, and 36h is the 7th group, uses small animal imaging instrument to observe FITC labelling crosslinked microsphere
Distribution situation in mouse GI tract.
Distribution results in FITC fluorescent-labeled microspheres mice digestive tract is analyzed as shown in Figure 3.
Respond control-release microsphere distribution situation in mice digestive tract to investigate the pH of the present invention further, fill to mice
Stomach FITC labelling chitosan/calcium alginate control-release microsphere, different time points after gavage (2,4,6,12,16,24,36h) see
Examine microsphere distribution situation in gastrointestinal tract, to observe the positioning scenarios of control-release microsphere.Can from the fluorescence intensity photo of Fig. 3
Going out, front 12h microsphere is mainly gathered in stomach, and from 16h, microsphere starts to be collected in a large number colon, gathers at most during 24h.
Result shows that colon positioning release microsphere prepared by the present invention can be in being administered 16-36h, by icariin drug accumulation at knot
Intestines position, thus reach the purpose of magnetic target therapy colitis.
The pharmacodynamic evaluation analysis of embodiment 6 icariin/chitosan/calcium alginate control-release microsphere
1, the foundation of the alcohol induced colitis model of TNBS/
Rats with ulcerative colitis alcohol induced for TNBS/ is classical Ulcerative Colitis Model.Choosing is healthy male
Property SD rat, after fasting 24h, by the chloral hydrate anesthesia of 0.3mL/100g lumbar injection 10%, lies on the back and is fixed on rat experiment
On plate, by gastric perfusion needle by the deep about 8cm of the light and slow insertion of anus, push 5%TNBS/ ethanol by 100mg/kg dosage (i.e. 4mL/kg)
Solution.Keep anus high-order 5 minutes after pouring into colon, prevent medicine from flowing out, with putting back in cage by rat, treat that nature is revived.
2, trial drug
Icariin/chitosan/calcium alginate the control-release microsphere of the embodiment of the present invention 2 preparation；
Positive drug: dexamethasone (tablet), Tianjin Lisheng Pharmaceutical Co., Ltd.'s (0.75mg/ sheet, totally 100)
Positive drug: icariin extract (purity > 99%): this Ai Me institute of Chinese materia medica is replaced in Nanjing
3, it is grouped and is administered
Modeling success enteritis rat is randomly divided into 7 groups, often group 8, is respectively blank group (Control), model group
(Model), positive drug group dexamethasone (DEX, 0.2mg/kg), low dose group (Low-dose, 30mg icariin microsphere/
Kg), middle dosage group (Medium-dose, 60mg icariin microsphere/kg), high dose group (High-dose, 90mg icariin
Microsphere/kg), icariin group (50mg icariin/kg).Starting gastric infusion after modeling success 24h, blank group gavage gives
Normal saline, model group gavage gives normal saline, and positive drug group gavage gives dexamethasone (0.2mg/kg), icariin group
Gavage gives icariin microsphere, low dose group (Low-dose, 30mg icariin microsphere/kg), middle dosage group (Medium-
Dose, 60mg icariin microsphere/kg), high dose group (High-dose, 90mg icariin microsphere/kg), icariin group
Gavage gives icariin (50mg icariin/kg), successive administration 6 days, and rat is put to death after being administered 24h by last, draws materials.
Rat oral gavage dosage with reference to " herbal pharmacology research methodology ", is given daily dosage with adult and is multiplied by rat conversion system
Number.4, draw materials and sample process
Rat Fast 24h before drawing materials, 10% chloral hydrate is lain on the back fixing after intraperitoneal injection of anesthesia.Abdomen is cut along ventrimeson
Wall, exposes abdominal tissue, careful separation colon (from ileocecus to anus mouth) to the open air.Cut off colon, longitudinally cut intestinal segment, ice open along mesentery
After normal saline flushing, perusal colonic mucosa form, take entire colon intestinal segment, absorb excessive moisture with filter paper, lie on
On glass plate, observing colon outward appearance, observing rapidly after cutting colon intestinal lumen has congestive content, checks that intestinal mucosa is with or without hyperemia
Edema, erosion and ulcer, record Injured colonic mucosa degree is also taken a picture, is weighed, residue colonic segment is sub-packed in sample and freezes
Depositing in pipe, in liquid nitrogen, quick-freezing is transferred to-80 DEG C of preservations stand-by (operation of drawing materials all is carried out on ice) in time.
5, pharmacodynamic evaluation result
5.1 Injured colonic mucosa indexes (CMDI)
Take rat anus to the colonic segment of cap end, cleaning mesentery and adhesion organization, cut off along the mesentery longitudinal axis,
Glacial phosphoric acid salt buffer rinses enteral remnants feces repeatedly, clean after be laid on filter paper to suck moisture, and observe big immediately
Body mucosa injury degree.
CMDI grade form is as shown in table 1.
Table 1 CMDI grade form
The measurement result of Injured colonic mucosa index (CMDI) is as shown in table 2.
Table 2 icariin/chitosan/calcium alginate control-release microsphere affects result to colitis model rat CMDI's
Colitis model Colonic Mucosa damage index test result indicate that, compares with blank group, and model group colon glues
Membrane damage is more serious, and CMDI value is higher, and administration group corresponding CMDI value is lowered with significance all occur, positive drug ground plug rice
Pine also is able to lower Colitis rat CMDI value, is administered high dose group and is better than positive drug group；Positive drug icariin also can
Enough lower Colitis rat CMDI value, be administered high dose group and be better than positive drug group.Control-release microsphere of the present invention is excessive with positive drug
Sheep icariin matched group is compared, and effectively reduces the using dosage of medicine, medicine can be made to arrive colon by control-release microsphere simultaneously
Position, medicine at colon site sustained release, and can be orally administered to icariin medicine more difficult arrival colon site, and controlled release is micro-
Ball can not only realize targeted therapy, also improves the bioavailability of medicine, more effectively reaches therapeutic effect.
5.2 icariin/chitosan/calcium alginate control-release microsphere is to scorching in Ulcerative Colitis Model rat colon tissue
Property correlation factor secretion impact
Protein lysate is used to carry out protein extraction in colon sample, with the BCA determination of protein concentration test kit (U.S.
Pierce company) measure protein content；ELISA method is used to measure IL-1 β and the secretion of TNF-α in colon's sample；IL-1β
Concentration use Rat IL-1 β ELISA kit (PeproTech company of the U.S.) be measured；The concentration of TNF-α uses Rat
TNF-α ELISA kit (eBioscience company of the U.S.) is measured, and specific experiment step is grasped according to test kit description
Make.
Ulcerative Colitis Model rat colon tissue IL-1 β is contained by icariin/chitosan/calcium alginate control-release microsphere
That measures affects measurement result as shown in table 3, Fig. 4；Icariin chitosan/calcium alginate control-release microsphere is to ulcerative colitis mould
The impact of type rat colon tissue T NF-alpha content is as shown in table 3, Fig. 5.
Table 3. icariin/chitosan/calcium alginate control-release microsphere is to Ulcerative Colitis Model rat colon tissue IL-
The impact of 1 β content
IL-1 β mainly mediates the generation of ulcerative colitis initial stage inflammation, and inflammatory cell can be promoted to enter colon
Internal lesion locations.By table 2 and Fig. 4 it can be seen that compare with blank group, the IL-1 β in model group colon is significantly raised,
There is pole significant difference (P < 0.01)；Positive drug dexamethasone matched group and microsphere drug treatment group all can significantly inhibit IL-1
The generation (P < 0.01 or P < 0.05) of β, wherein in medicine carrying microballoons, dosage group has in the generation of suppression Colitis rat IL-1 β
Pole significant difference (P < 0.01).Positive drug icariin matched group and microsphere drug treatment group all can significantly inhibit IL-1 β
Generation (P < 0.01 or P < 0.05), the control-release microsphere of the present invention effectively reduces the using dosage of medicine, can make medicine simultaneously
Thing arrives colon site by control-release microsphere, and medicine at colon site sustained release, and can be orally administered to icariin medicine
More difficult arrival colon site, control-release microsphere can not only realize targeted therapy, also improve the bioavailability of medicine, more effectively reach
To therapeutic effect.
Table 4. icariin chitosan/calcium alginate control-release microsphere is to Ulcerative Colitis Model rat colon tissue T NF-
The impact of alpha content
TNF-α mainly starts inflammatory factor, and mononuclear phagocyte and neutrophilic granulocyte etc. can be stimulated to synthesize other inflammatories
Cytokine.From table 4 and Fig. 5, comparing with blank group, model group TNF-α content is significantly raised, has pole significant difference
(P<0.01)；Compared with model group, positive drug dexamethasone matched group and medicine carrying microballoons high, medium and low dosage group TNF-α content
All significantly reducing (P < 0.05), medicine carrying microballoons high dose group inhibition is the most obvious；Positive drug icariin matched group and load
Medicine microsphere high, medium and low dosage group TNF-α content all significantly reduces (P < 0.05), and medicine carrying microballoons high dose group inhibition is the brightest
Aobvious.Effectively reducing the using dosage of medicine, medicine can be made simultaneously to pass through control-release microsphere and arrive colon site, medicine can be
Colon site sustained release, and it is orally administered to icariin medicine more difficult arrival colon site, control-release microsphere can not only realize
Targeted therapy, also improves the bioavailability of medicine, more effectively reaches therapeutic effect.
Claims (10)
1. the preparation method of an icariin control-release microsphere, it is characterised in that include the step that order below is carried out:
1) in sodium alginate soln, add calcium carbonate, icariin, stirring, make calcium carbonate, icariin be suspended in alginic acid
In sodium solution, prepare calcium carbonate-icariin-sodium alginate suspension；
2) emulsifying agent is joined in liquid paraffin, stirring, mixing, make emulsifying agent-paraffin solution；
3) under stirring, calcium carbonate-icariin-sodium alginate suspension is mixed with emulsifying agent-paraffin solution, carries out
Emulsifying, forms emulsification system；It is subsequently added into acid initiator, carries out acid initiation and process, generate icariin-calcium alginate micro-
Ball；
4) icariin-calcium alginate microsphere is joined in film forming chitosan solution, mix homogeneously, carry out multiple electrolyte complexation
Reaction, chitosan is wrapped in the surface of icariin-calcium alginate microsphere, makes chitosan-icariin-calcium alginate micro-
Ball；
5) chitosan-icariin-calcium alginate microsphere is mixed with cross-linking agent, carry out cross-linking reaction, obtain icariin control
Release microsphere.
Preparation method the most according to claim 1, it is characterised in that step 1) described in the weight of icariin and Sargassum
The ratio of the volume of acid sodium solution is 1-3:100；The concentration of described sodium alginate soln is 0.5-2% (w/v).
Preparation method the most according to claim 1 and 2, it is characterised in that step 2) described in emulsifying agent selected from sorbester p17
Or the one in Tween 80 or two kinds.
Preparation method the most according to claim 1 and 2, it is characterised in that step 2) described in emulsifying agent and liquid paraffin
The ratio of volume be 0.5-2:100.
Preparation method the most according to claim 1 and 2, it is characterised in that step 3) described in emulsifying temperature be 35-
45℃；The emulsifying time is 30-60min.
Preparation method the most according to claim 1 and 2, it is characterised in that step 3) described in acid initiator select ice vinegar
Acid solution.
Preparation method the most according to claim 1 and 2, it is characterised in that step 4) described in film forming chitosan solution be
Chitosan-acetum.
Preparation method the most according to claim 1 and 2, it is characterised in that step 5) described in the temperature < 10 of cross-linking reaction
℃；Cross-linking reaction time is 1.5-3h.
9. an icariin control-release microsphere, it is characterised in that be prepared from according to method as described in claim 1-8 is arbitrary.
10. the icariin control-release microsphere as claimed in claim 9 application in preparation treatment colonic diseases.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109528683A (en) * | 2018-12-13 | 2019-03-29 | 浙江华康药业股份有限公司 | A kind of arabinose fixed point release produces the regulation method of butyric to enteral |
| CN114099473A (en) * | 2021-11-30 | 2022-03-01 | 华臻 | Slow release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells and preparation thereof |
| CN115068494A (en) * | 2022-08-22 | 2022-09-20 | 广东工业大学 | Application of icariin in preparation of medicine for treating irritable bowel syndrome |
-
2016
- 2016-08-19 CN CN201610701701.7A patent/CN106309380A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| JIE ZHOU ETAL: "A preliminary study of crosslinked alginate-chitosan microshperes for delivery system", 《ADVANCED MATERIALS RESEARCH》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109528683A (en) * | 2018-12-13 | 2019-03-29 | 浙江华康药业股份有限公司 | A kind of arabinose fixed point release produces the regulation method of butyric to enteral |
| CN114099473A (en) * | 2021-11-30 | 2022-03-01 | 华臻 | Slow release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells and preparation thereof |
| CN115068494A (en) * | 2022-08-22 | 2022-09-20 | 广东工业大学 | Application of icariin in preparation of medicine for treating irritable bowel syndrome |
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