CN109884223A - A kind of strychnia solubility micropin, preparation method and detection method and application - Google Patents
A kind of strychnia solubility micropin, preparation method and detection method and application Download PDFInfo
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- CN109884223A CN109884223A CN201910134480.3A CN201910134480A CN109884223A CN 109884223 A CN109884223 A CN 109884223A CN 201910134480 A CN201910134480 A CN 201910134480A CN 109884223 A CN109884223 A CN 109884223A
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Abstract
The present invention relates to a kind of strychnia solubility micropins, by 0.012002 part~0.012010 part of strychnia, 0.998 part~1.006 parts of chondroitin sulfate, 0.997 part~1.005 parts of polyvinylpyrrolidone compositions, the present invention is that soluble micropin is prepared using vacuum decompression method, using strychnia content in high effective liquid chromatography for measuring sample and transdermal test in vitro effect, the strychnia solubility micropin morphology and satisfactory mechanical property of preparation, solve the problems, such as that drug is difficult to through cuticula, it is very close to solve strychnia effective dose and toxic dose in terms for the treatment of rheumatoid arthritis disease, the problem of seriously limiting its application clinically；Strychnia solubility micropin prepared by the present invention can significantly mitigate ankle-joint position synovial membrane epithelial proliferation and cartilage erosion, maintain joint space, have good therapeutic effect to rat RA；The strychnia content assaying method of foundation is simply accurate, and Feasible degree is high.
Description
Technical field
The present invention relates to field of medicine invention, and in particular to a kind of strychnia solubility micropin, preparation method and detection
Methods and applications.
Background technique
Rheumatoid arthritis (RA) is today's society to one of maximum disease of anthropogenic influence, has disability rate high (only secondary
In diseases such as malaria), disease incidence high (being averagely about 0.5%~1.0% in the whole world), high recurrence rate the features such as, and it is current
Drug side-effect is big, and the drug and method of the special efficacy for the treatment of RA there is no therefore to study a kind for the treatment of class wind of high-efficiency low-toxicity so far
The drug of wet arthritis is today's society urgent problem to be solved.
Have scholar research shows that strychnia (Bru) treatment aspect of inflammation have significant curative effect, be treat RA a kind of reason
Think drug, has good inhibiting effect to the proliferation of rheumatoid arthritis fibroblast-like synoviocyte.However, Bru has poison
Property, although toxicity is only the 1/20 of vomiting nut, tissue affinity is high, can be distributed into tissue to each device rapidly after administration
Official can especially penetrate the toxicity of initiation cental system after blood-brain barrier, seriously can cause death when poisoning.
Percutaneous drug administration preparation research [D] the Nanjing University of Traditional Chinese Medicine of Wang Xuan optimization strychnia total alkaloid, 2012
(04) during reporting percutaneous dosing, Central Analgesic Effect may also can be reduced while reducing maincenter toxicity, but to periphery
The influence of analgesic activity is minimum, therefore percutaneous dosing is the promising approach for making strychnia Synergy and attenuation.
Hu Wei, Chen Jun, Cai Baochang wait analgesic activity [J] the China traditional Chinese medicine academic periodical of Brucine by Transdermal Administration,
2008,26 (2): it can quickly be eliminated after 385-386 but Bru percutaneous absorbtion, half-life period (t1/2) short, pole is unfavorable for its performance
Drug effect.Toxicity is big and half-life short this two major features limit the application of strychnia clinically.
Wu Yu waits the transdermal test in vitro of strychnia carrier and promotees infiltration property research [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA, and 2016,
41 (16): 3009-3015 has researcher to develop Bru at patch, micro emulsion, vesica and liposome.
The problem of percutaneous dosing can extend the release time of drug, overcome half-life short, however this layer of barrier of cuticula
Still prevent some drugs through skin, it is difficult to reach efficient purpose always.
Microneedle array is a kind of emerging percutaneous dosing technology, it can pierce through keratoderma without touching pain mind
Patient compliance can be improved.Micropin can be divided into hollow microneedles and solid microneedles two major classes, and hollow microneedles, which are equivalent to, lines up array
Injection needle, drug is released out of each needle tubing to playing drug effect after being pierced into skin.Solid microneedles can be divided into biology again
Degradability micropin, skin pretreatment micropin and 3 seed type of medication coat micropin.
Present invention design uses high molecular material chondroitin sulfate and polyvinylpyrrolidone to subtract for matrix and using vacuum
Strychnia is prepared into biodegradable micropin by platen press, using strychnia content in high effective liquid chromatography for measuring sample and
Transdermal test in vitro effect, the strychnia solubility micropin morphology and satisfactory mechanical property of preparation, solves drug and is difficult to penetrate
The problem of cuticula, solving strychnia, effective dose and toxic dose be very in terms for the treatment of rheumatoid arthritis disease
It approaches, seriously limit the problem of its application clinically；Strychnia solubility micropin prepared by the present invention can be significant
Mitigate ankle-joint position synovial membrane epithelial proliferation and cartilage erosion, maintain joint space, there is good therapeutic effect to rat RA；
The strychnia content assaying method of foundation is simply accurate, and Feasible degree is high.
Summary of the invention
The purpose of the invention is to provide a kind of strychnia solubility micropin.
It is another object of the present invention in order to provide a kind of preparation method of strychnia solubility micropin.
It is another object of the present invention in order to provide a kind of detection method of strychnia solubility micropin；
It is another object of the present invention in order to provide a kind of strychnia solubility micropin in treatment rheumatoid arthritis
Application in terms of disease.
Strychnia solubility micropin of the present invention is made according to the following ratio of following component: strychnia
0.012002 part~0.012010 part, 0.998 part~1.006 parts of chondroitin sulfate, 0.997 part of polyvinylpyrrolidone~
1.005 part.
Preferably, strychnia solubility micropin of the present invention is made according to the following ratio of following component: vomiting nut
0.012004 part~0.012008 part of alkali, 1.000 parts~1.004 parts of chondroitin sulfate, 0.999 part of polyvinylpyrrolidone~
1.003 part.
It is further preferred that strychnia solubility micropin of the present invention is made according to the following ratio of following component:
0.012006 part of strychnia, 1.002 parts of chondroitin sulfate, 1.001 parts of polyvinylpyrrolidone.
The preparation method of strychnia solubility micropin of the present invention is made of following steps:
1) strychnia of formula ratio is weighed, 1.4ml~1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia
Pharmaceutical aqueous solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h~4h, is put into 18 DEG C~22 DEG C vacuum ovens, is depressurized；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days~5 days, demoulding, obtaining strychnia can
Dissolubility micropin.
Preferably, the preparation method of strychnia solubility micropin of the present invention is made of following steps:
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, is depressurized；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Aforesaid vacuum condition is 0.07MPa~0.09MPa.
The aforesaid vacuum time is 25min~35min.
Strychnia solubility micropin measuring method of the present invention, according to high effective liquid chromatography for measuring, specific method
Are as follows:
(1) chromatographic condition: Agilent C18 4.6mm × 250mm, 5um, mobile phase A: Mobile phase B is by volume
30:70, mobile phase A are methanol, and Mobile phase B is water 230 by volume: acetic acid 2.4: triethylamine 0.3, Detection wavelength 265nm,
Flow velocity 1ml/min, 30 DEG C of column temperature；
(2) preparation of solution
1) preparation of reference substance solution
Strychnia reference substance 0.00206g is weighed, it is accurately weighed, it is placed in 25ml volumetric flask, methanol is added to dissolve and determine
Hold, the strychnia reference substance solution that concentration is 80 μ g/ml is made；
2) preparation of test solution
Precision weighs strychnia solubility micropin 0.1g, is placed in 25ml volumetric flask, add 25ml methanol power 200w,
45min~50min is ultrasonically treated under frequency 50kHz, the structure for destroying micropin makes to contain sufficiently to be released in drug therein,
After constant volume through the filtration of 0.22 μm of miillpore filter to get；
3) preparation of blank micropin solution
Precision weighs blank micropin 0.1g, is placed in 25ml volumetric flask, adds 25ml methanol in power 200w, frequency 50kHz
Lower ultrasonic treatment 45min~50min, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, is passed through after constant volume
0.22 μm of miillpore filter filtration is to get blank micropin solution；
(3) it measures
1) specificity measurement takes the reference substance solution under Section 2, test solution, and each 10 μ l of blank micropin solution is pressed
Chromatographic condition sample introduction under Section 1, measurement；
2) linear relationship measures
Precision draws strychnia reference substance solution 0.5 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under Section 2, by the
(1) chromatographic condition sample introduction under item, measurement；
3) precision measures
Precision draws strychnia reference substance solution 2.0ml under Section 2, is settled to 5ml with mobile phase, obtaining concentration is
The strychnia reference substance solution of 0.032 μ g/ μ l, precision draw 10 μ l sample introductions, by chromatographic condition sample introduction under Section 1, survey
It is fixed；
4) repeatability measurement
It weighs with a collection of strychnia solubility micropin, is placed in 25ml volumetric flask, adds methanol to dissolve and be settled to scale,
Precision draws 10 μ l, by chromatographic condition sample introduction under Section 1, measurement；
5) Stability Determination
Precision is drawn with a 10 μ l of strychnia test solution in 0h, 2h, 4h, 6h, 8h, 12h, and for 24 hours, 48h is by the
(1) chromatographic condition sample introduction under item, measurement；
6) measurement of the rate of recovery
Appropriate strychnia solubility micropin powder is weighed, is added in the ratio that sample size and reference substance content are 1:1
Reference substance, precision are drawn 10 μ l and are measured by chromatographic condition under Section 1；
7) sample size measures
Take the reference substance solution under Section 2, test solution, each 10 μ l of blank micropin solution, by color under Section 1
Spectral condition sample introduction measures strychnia content in sample.
The preparation method of blank micropin of the present invention is the following steps are included: weigh polyvinylpyrrolidone 0.500g, sulphur
Aching and limp ossein 0.500g is added 0.7ml~0.9ml pure water, is stirred evenly rapidly with glass bar, is cast in after being swollen 2h~4h
It on micropin mold, is placed in 18 DEG C~22 DEG C of vacuum oven, is evacuated to 0.05MPa~0.09MPa, decompression 25min~
35min, taking-up are placed in 2 DEG C~6 DEG C refrigerators, are stood overnight defoaming and are moved back to drier solidification 3~5 days, demould to obtain the final product.
Application of the strychnia solubility micropin of the present invention in terms for the treatment of rheumatoid arthritis disease.
Polyvinylpyrrolidone of the present invention can be in PVP K30, polyvinylpyrrolidone K25, poly- second
It chooses any one kind of them in alkene pyrrolidone K90, preferably PVP K30.
Preferably one block of non-woven fabrics is taken directly to be laid in the stromal surface after depressurizing after depressurizing in preparation method of the present invention,
Being placed in drier solidifies micropin slowly.
The invention has the advantages that:
1, the present invention is host material using polyvinylpyrrolidone and chondroitin sulfate, takes vacuum decompression method, is prepared
Strychnia solubility micropin morphology and satisfactory mechanical property.
2, strychnia is prepared into biodegradable micropin, not only acts as slow releasing function, patient need not repeatedly dressing, mention
The high compliance of patient, solves the problems, such as half-life short, moreover it is possible to penetrate cuticula, drug release is promoted to play drug effect, solution
Drug is difficult to the problem through cuticula.
3, using strychnia content in high effective liquid chromatography for measuring sample and transdermal test in vitro effect, the line of strychnia
Property regression equation be Y=2E+06x+11263 (R2=0.9997), the range of linearity is 0.04 μ of μ g~0.8 g；Strychnia is solvable
Property micropin 48 hours in the unit area transdermal penetration amount of micropin processing group be 7.9406 times without micropin processing group.
4, the strychnia content assaying method that the present invention establishes is simply accurate, and Feasible degree is high.
5, the present invention has searched out the material that matter is soft, nonirritant by test, due to taking vacuum decompression method and subtracting
One block of non-woven fabrics is taken directly to be laid in the stromal surface after depressurizing after pressure, the dry sequence of matrix is slowly reached by pin hole
Back sheet, micropin prepared by the present invention have reached the soft form of needle dura mater, make patient to can move freely since the medicine.
6, the preparation process of strychnia solubility micropin and supplementary material are screened by stringent science, significant in efficacy,
It has no toxic side effect, solves strychnia effective dose and denaturant in terms for the treatment of rheumatoid arthritis disease
Measure it is very close, seriously limit the problem of its application clinically.
7, strychnia solubility micropin prepared by the present invention can significantly mitigate rat articular swelling, significantly reduce serum
The level of middle TNF- ɑ and IL-1 β mitigate ankle-joint position synovial membrane epithelial proliferation and cartilage erosion, joint space are maintained, to rat
RA has good therapeutic effect.
8, the present invention is proved by safety testing, and safe without toxic side effect is used in dosage range.
Detailed description of the invention:
Fig. 1 strychnia solubility micropin preparation flow figure
Fig. 2 strychnia solubility micropin morphology investigates figure；A: microscope top view image；B. strychnia solubility micropin
Enlarged drawing；C. microscope side view
Fig. 3 strychnia solubility micropin puncture test figure；A. aluminium foil punctures experiment；B. through methylene blue solution dyeing
Skin penetrating experiment；C. skin penetrating tests audio-visual picture
Strychnia reference substance HPLC chromatogram in the test of Fig. 4 specificity
Strychnia solubility micropin HPLC chromatogram in the test of Fig. 5 specificity
Strychnia feminine gender HPLC chromatogram is removed in the test of Fig. 6 specificity
Fig. 7 strychnia solubility micropin Ligustrazine hydrochloride trial curve figure a. micropin punctures skin group；B. micropin is molten
Liquid penetrating absorption group (skin is without micropin processing)
Specific embodiment
Below by specific embodiment, technical solution of the present invention is further illustrated.
Embodiment 1
The preparation method of blank micropin:
Polyvinylpyrrolidone 0.500g, chondroitin sulfate 0.500g are weighed, 0.7ml pure water is added, it is fast with glass bar
Speed stirs evenly, and is cast on micropin mold after being swollen 2h, is placed in 18 DEG C of vacuum oven, is evacuated to 0.05MPa, subtract
Press 25min, taking-up be placed in 2 DEG C of refrigerators, stand overnight defoaming move back to drier solidify 3 days, demoulding to get.
Embodiment 2
The preparation method of blank micropin:
Polyvinylpyrrolidone 0.500g, chondroitin sulfate 0.500g are weighed, 0.9ml pure water is added, it is fast with glass bar
Speed stirs evenly, and is cast on micropin mold after being swollen 4h, is placed in 22 DEG C of vacuum oven, is evacuated to 0.09MPa, subtract
Press 35min, taking-up be placed in 6 DEG C of refrigerators, stand overnight defoaming move back to drier solidify 5 days, demoulding to get.
Embodiment 3
The preparation method of blank micropin:
Polyvinylpyrrolidone 0.500g, chondroitin sulfate 0.500g are weighed, 0.8ml pure water is added, it is fast with glass bar
Speed stirs evenly, and is cast on micropin mold after being swollen 3h, is placed in 20 DEG C of vacuum oven, is evacuated to 0.07MPa, subtract
Press 30min, taking-up be placed in 4 DEG C of refrigerators, stand overnight defoaming move back to drier solidify 4 days, demoulding to get.
Embodiment 4
Strychnia solubility micropin
Ingredient: strychnia 0.012002g, chondroitin sulfate 0.998g, polyvinylpyrrolidone 0.997g.
Preparation method:
1) strychnia of formula ratio is weighed, 1.4ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h, is put into 18 DEG C of vacuum ovens, is depressurized under the conditions of 0.07MPa
25min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 5
Strychnia solubility micropin
Ingredient: strychnia 0.012002g, chondroitin sulfate 0.998g, polyvinylpyrrolidone 0.997g.
1) strychnia of formula ratio is weighed, 1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h~4h, is put into 22 DEG C of vacuum ovens, is depressurized under the conditions of 0.09MPa
35min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 5 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 6
Strychnia solubility micropin
Ingredient: strychnia 0.012002g, chondroitin sulfate 0.998g, polyvinylpyrrolidone 0.997g.
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, 30min is depressurized under the conditions of 0.08MPa；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 7
Strychnia solubility micropin
Ingredient: strychnia 0.012010g, chondroitin sulfate 1.006g, polyvinylpyrrolidone 1.005g.
Preparation method:
1) strychnia of formula ratio is weighed, 1.4ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h, is put into 18 DEG C of vacuum ovens, is depressurized under the conditions of 0.07MPa
25min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 8
Strychnia solubility micropin
Ingredient: strychnia 0.012010g, chondroitin sulfate 1.006g, polyvinylpyrrolidone 1.005g.
1) strychnia of formula ratio is weighed, 1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 4h, is put into 22 DEG C of vacuum ovens, is depressurized under the conditions of 0.09MPa
35min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 5 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 9
Strychnia solubility micropin
Ingredient: strychnia 0.012010g, chondroitin sulfate 1.006g, polyvinylpyrrolidone 1.005g.
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, 30min is depressurized under the conditions of 0.08MPa；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 10
Strychnia solubility micropin
Ingredient: strychnia 0.012004g, chondroitin sulfate 1.000g, polyvinylpyrrolidone 0.999g.
Preparation method:
1) strychnia of formula ratio is weighed, 1.4ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h, is put into 18 DEG C of vacuum ovens, is depressurized under the conditions of 0.07MPa
25min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 11
Strychnia solubility micropin
Ingredient: strychnia 0.012004g, chondroitin sulfate 1.000g, polyvinylpyrrolidone 0.999g.
1) strychnia of formula ratio is weighed, 1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h~4h, is put into 22 DEG C of vacuum ovens, is depressurized under the conditions of 0.09MPa
35min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 5 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 12
Strychnia solubility micropin
Ingredient: strychnia 0.012004g, chondroitin sulfate 1.000g, polyvinylpyrrolidone 0.999g.
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, 30min is depressurized under the conditions of 0.08MPa；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 13
Strychnia solubility micropin
Ingredient: strychnia 0.012008g, chondroitin sulfate 1.004g, polyvinylpyrrolidone 1.003g.
Preparation method:
1) strychnia of formula ratio is weighed, 1.4ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h, is put into 18 DEG C of vacuum ovens, is depressurized under the conditions of 0.07MPa
25min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 14
Strychnia solubility micropin
Ingredient: strychnia 0.012008g, chondroitin sulfate 1.004g, polyvinylpyrrolidone 1.003g.
1) strychnia of formula ratio is weighed, 1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 4h, is put into 22 DEG C of vacuum ovens, is depressurized under the conditions of 0.09MPa
35min；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 5 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 15
Strychnia solubility micropin
Ingredient: strychnia 0.012008g, chondroitin sulfate 1.004g, polyvinylpyrrolidone 1.003g.
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, 30min is depressurized under the conditions of 0.08MPa；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 16
Strychnia solubility micropin
Ingredient: strychnia 0.012006g, chondroitin sulfate 1.002g, polyvinylpyrrolidone 1.001g.
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug water
Solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia drug water is added after mixing in chondroitin sulfate
In solution, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, 30min is depressurized under the conditions of 0.07MPa；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, it is micro- to obtain strychnia solubility
Needle.
Using: treatment rheumatoid arthritis disease.
Embodiment 4- embodiment 16 is measured by the measuring method of following any embodiment
Embodiment 17: measuring method (one):
(1) chromatographic condition: Agilent C18 4.6mm × 250mm, 5um, mobile phase A: Mobile phase B is by volume
30:70, mobile phase A are methanol, and Mobile phase B is water 230 by volume: acetic acid 2.4: triethylamine 0.3, Detection wavelength 265nm,
Flow velocity 1ml/min, 30 DEG C of column temperature；
(2) preparation of solution
1) preparation of reference substance solution
Strychnia reference substance 0.00206g is weighed, it is accurately weighed, it is placed in 25ml volumetric flask, methanol is added to dissolve and determine
Hold, the strychnia reference substance solution that concentration is 80 μ g/ml is made；
2) preparation of test solution
Precision weighs strychnia solubility micropin 0.1g, is placed in 25ml volumetric flask, add 25ml methanol power 200w,
It is ultrasonically treated 45min under frequency 50kHz, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, after constant volume
Through the filtration of 0.22 μm of miillpore filter to get；
3) preparation of blank micropin solution
Precision weighs blank micropin 0.1g, is placed in 25ml volumetric flask, adds 25ml methanol in power 200w, frequency 50kHz
Lower ultrasonic treatment 45min, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, micro- through 0.22 μm after constant volume
Hole filter membrane filters to get blank micropin solution；
(3) it measures
1) specificity measurement takes the reference substance solution under Section 2, test solution, and each 10 μ l of blank micropin solution is pressed
Chromatographic condition sample introduction under Section 1, measurement；
2) linear relationship measures
Precision draws strychnia reference substance solution 0.5 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under Section 2, by the
(1) chromatographic condition sample introduction under item, measurement；
3) precision measures
Precision draws strychnia reference substance solution 2.0ml under Section 2, is settled to 5ml with mobile phase, obtaining concentration is
The strychnia reference substance solution of 0.032 μ g/ μ l, precision draw 10 μ l sample introductions, by chromatographic condition sample introduction under Section 1, survey
It is fixed；
4) repeatability measurement
It weighs with a collection of strychnia solubility micropin, is placed in 25ml volumetric flask, adds methanol to dissolve and be settled to scale,
Precision draws 10 μ l, by chromatographic condition sample introduction under Section 1, measurement；
5) Stability Determination
Precision is drawn with a 10 μ l of strychnia test solution in 0h, 2h, 4h, 6h, 8h, 12h, and for 24 hours, 48h is by the
(1) chromatographic condition sample introduction under item, measurement；
6) measurement of the rate of recovery
Appropriate strychnia solubility micropin powder is weighed, is added in the ratio that sample size and reference substance content are 1:1
Reference substance, precision are drawn 10 μ l and are measured by chromatographic condition under Section 1；
7) sample size measures
Take the reference substance solution under Section 2, test solution, each 10 μ l of blank micropin solution, by color under Section 1
Spectral condition sample introduction measures strychnia content in sample.
Embodiment 18: measuring method (two):
(1) chromatographic condition: Agilent C18 4.6mm × 250mm, 5um, mobile phase A: Mobile phase B is by volume
30:70, mobile phase A are methanol, and Mobile phase B is water 230 by volume: acetic acid 2.4: triethylamine 0.3, Detection wavelength 265nm,
Flow velocity 1ml/min, 30 DEG C of column temperature；
(2) preparation of solution
1) preparation of reference substance solution
Strychnia reference substance 0.00206g is weighed, it is accurately weighed, it is placed in 25ml volumetric flask, methanol is added to dissolve and determine
Hold, the strychnia reference substance solution that concentration is 80 μ g/ml is made；
2) preparation of test solution
Precision weighs strychnia solubility micropin 0.1g, is placed in 25ml volumetric flask, add 25ml methanol power 200w,
It is ultrasonically treated 50min under frequency 50kHz, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, after constant volume
Through the filtration of 0.22 μm of miillpore filter to get；
3) preparation of blank micropin solution
Precision weighs blank micropin 0.1g, is placed in 25ml volumetric flask, adds 25ml methanol in power 200w, frequency 50kHz
Lower ultrasonic treatment 50min, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, micro- through 0.22 μm after constant volume
Hole filter membrane filters to get blank micropin solution；
(3) it measures
1) specificity measurement takes the reference substance solution under Section 2, test solution, and each 10 μ l of blank micropin solution is pressed
Chromatographic condition sample introduction under Section 1, measurement；
2) linear relationship measures
Precision draws strychnia reference substance solution 0.5 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under Section 2, by the
(1) chromatographic condition sample introduction under item, measurement；
3) precision measures
Precision draws strychnia reference substance solution 2.0ml under Section 2, is settled to 5ml with mobile phase, obtaining concentration is
The strychnia reference substance solution of 0.032 μ g/ μ l, precision draw 10 μ l sample introductions, by chromatographic condition sample introduction under Section 1, survey
It is fixed；
4) repeatability measurement
It weighs with a collection of strychnia solubility micropin, is placed in 25ml volumetric flask, adds methanol to dissolve and be settled to scale,
Precision draws 10 μ l, by chromatographic condition sample introduction under Section 1, measurement；
5) Stability Determination
Precision is drawn with a 10 μ l of strychnia test solution in 0h, 2h, 4h, 6h, 8h, 12h, and for 24 hours, 48h is by the
(1) chromatographic condition sample introduction under item, measurement；
6) measurement of the rate of recovery
Appropriate strychnia solubility micropin powder is weighed, is added in the ratio that sample size and reference substance content are 1:1
Reference substance, precision are drawn 10 μ l and are measured by chromatographic condition under Section 1；
7) sample size measures
Take the reference substance solution under Section 2, test solution, each 10 μ l of blank micropin solution, by color under Section 1
Spectral condition sample introduction measures strychnia content in sample.
In order to further verify feasibility of the invention, inventor has carried out a series of test, specific as follows:
1 instrument, reagent and reagent
1.1 instrument
DZF-6050 type vacuum oven (Shanghai Qi Xin scientific instrument Co., Ltd), HF-50 digital display tie up tensiometer (Foochow
Ai Pu Instrument Ltd.), Shimadzu LC-2010 type high performance liquid chromatograph (Japanese Shimadzu Corporation), Agilent C18 chromatographic column,
Micropin mold, RYJ-12B type drug transdermal diffusion instrument (Shanghai Huanghai Sea medicine inspection Instrument Ltd.), SK8210HP type ultrasonic wave are clear
Wash instrument (Shanghai High Kudos Science Instrument Co., Ltd.), microscope (Shun's space perseverance level device), aluminium foil, ten a ten thousandth of JA2003 type point
Analyse balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.), one thousandth assay balance (SHIMADZU/ Shimadzu).
1.2 reagent
Strychnia reference substance (purity: 99.27%, quality inspection Bioisystech Co., Ltd, BeiJing ZhongKe, lot number:
, triethylamine (Tianjin section 17091910), methanol (Tianjin perseverance photochemical agent Manufacturing Co., Ltd, lot number: 20150307)
Mi Ou chemical reagent Co., Ltd, lot number: 20150120), isopropanol (Tianjin Zhi Yuan chemical reagent Co., Ltd, lot number:
20150901), acetic acid, 95% ethyl alcohol, pure water (Hangzhou Wahaha Group Co., Ltd), PVP K30 (Henan
Beaune Biotechnology Co., Ltd), chondroitin sulfate (Hunan Tian Feng Biotechnology Co., Ltd), methylene blue solution (Tianjin
Ke Miou chemical reagent Co., Ltd, city), physiological saline (Guizhou Kelun Pharmaceutical Co., Ltd.).
2 Study on Preparation
The preparation of 2.1 blank micropins
2.1.1 the selection of matrix
Influence of each matrix of table 1 to micropin mouldability
As known from Table 1, CS+PVP punctures that effect is good, good appearance, therefore as preferred substrate of the invention.
2.1.2 the influence of amount of water
Polyvinylpyrrolidone 0.500g is weighed, chondroitin sulfate 0.500g, equivalent weighs 6 parts, mixes respectively, with micropin
Mouldability is inspection target, is separately added into 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml pure water, is stirred rapidly with glass bar
Uniformly, it is cast on micropin mold after being swollen 3h, is placed in 20 DEG C of vacuum oven, is evacuated to 0.07MPa, depressurized
30min, taking-up be placed in refrigerator (4 DEG C, similarly hereinafter), stand overnight defoaming move back to drier solidification demould to obtain the final product.
The result shows that: amount of water is excessive lower than the when matrix viscosity of 0.7ml, and decompression process is not easy to eliminate after generating bubble,
It easily shrinks in drying process when amount of water is 0.9ml, is easy after generating bubble without viscosity, decompression process when amount of water when 0.8ml
It eliminates and drying process is not easy to shrink, therefore final determining amount of water is 0.7ml~0.9ml, amount of water preferred embodiment is
0.8ml, i.e. the 80% of matrix quality；Preferable scheme is 0.9ml, feasible program 0.7ml.
2.1.3 the influence of matrix ratios
Polyvinylpyrrolidone is weighed by table 2 respectively, chondroitin sulfate is uniformly mixed, the pure water of 0.48ml is added, and uses
Glass bar quickly stirs evenly, and is cast in micropin mold after being swollen 3h, is placed in 20 DEG C of vacuum ovens, is evacuated to
0.07MPa depressurizes 30min, and taking-up, which is placed in refrigerator and stands overnight defoaming and move back into drier, solidifies demoulding, obtains blank micropin.
In the form of micropin and effect is punctured as examination index, microscopically observation micropin mouldability, and is examined with aluminium foil puncture test
The mechanical performance of micropin is examined, is evaluated with " excellent " " good " two grades, as a result as shown in the table, the preferred substrate finally determined
Proportion is 1:1.
2 strychnia solubility micropin assistant ingredients of table are than investigating
2.2. preparation process screens explanation
2.2.1 this research is in the screening of strychnia solubility microneedle preparation method, initially use vacuum decompression method, knot
Fruit shows that negative pressure can make matrix infiltration into microporous, but glutinous due to polyvinylpyrrolidone and the host material of chondroitin sulfate preparation
Degree is big, and the gap left after matrix infiltration into microporous cannot be filled up to form bubble in time, then uses the low speed of horizontal rotor
The micropin of centrifuge (maximum speed 5000rpm) preparation also can smoothly make matrix infiltration into microporous, and not will form bubble, but due to
It is influenced simultaneously by centrifugal force and gravity in centrifugal process, matrix is easy to overflow mold, causes strychnia solubility microneedle patch
Sheet weight difference is big, matrix overflow it is excessive in the case where part when resulting even in centrifugation upward penetrated into without material to be formed it is micro-
Needle can not solve the problems, such as this increasing matrix viscosity.Later in moist ring after repetition test finds vacuum decompression
It is stood overnight under border, the bubble of formation can be eliminated automatically, this method as finally determined.
2.1.2 the appearance time of strychnia has moved back about 4min in assay experiment, and reason may be with climate change
It is related.
It 2.1.3, will be micro- the invention aims to find the material that matter is soft, nonirritant about the screening of back lining materials
Needle is prepared into the soft form of needle dura mater, so that patient once investigated carboxymethyl cellulose to that can move freely since the medicine
Sodium, carbomer, chitosan, chondroitin sulfate, gelatin etc. are used as back lining materials, and experimental method is that first injection molding decompression penetrates into matrix
After micropore, then the matrix on micropore upper layer is scraped off with metal spoon, the decompression after the back lining materials investigated, which is added, makes itself and micropin material
Material is close to, final curing demoulding.It was found that the number of micropin significantly reduces, aciculiform is also unintelligible complete, but due to matrix viscosity
It is excessive, the matrix in pin hole is easily affected during scraping off pin hole upper layer, causes the number of micropin to significantly reduce, aciculiform
It is unintelligible complete.In order to avoid the above problem, then takes one block of non-woven fabrics to be directly laid in the stromal surface after depressurizing, be placed in
Drier solidifies micropin slowly.As a result, it has been found that the dry sequence of matrix is that back sheet is slowly reached by pin hole, system of the present invention
Standby micropin has achieved the purpose that needle dura mater is soft.
The preparation of 2.3 strychnia solubility micropins
2.3.1 the determination of drug dose
He Xiaowei, Fan Xiaoping, Zhong Tao, wait strychnia novel Drug Delivery Systems treatment rheumatoid arthritis research into
[J] China traditional Chinese medicine academic periodical is opened up, 2015,33 (12): describes strychnia in 2908-2911 in the agent of 8~16mg/kg range
Amount has apparent inhibiting effect to inflammatory reaction, on immune organ weight almost without influence.Present invention determine that dosage be 8mg/
Kg, in terms of 180g rat, then every microneedle patch should contain 1.44mg strychnia.
2.3.2 the preparation flow of strychnia solubility micropin (since drug composition is the same, only chooses embodiment 16
Do corresponding test).
Strychnia 0.012006g is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, PVP1.001g is weighed,
CS1.002g is added in strychnia pharmaceutical aqueous solution after mixing, and quickly stirring makes to be formed uniform solution, be swollen after 3h casting with
Micropin mold (casting preparation six micropins), is put into 20 DEG C of vacuum ovens, and 30min is depressurized under the conditions of 0.07MPa, take out in
Interior stands overnight defoaming and moves back into drier solidification demoulding, obtains strychnia solubility micropin.Strychnia solubility is micro-
Needle preparation flow is shown in Fig. 1.
The main feature of 3 strychnia solubility micropins
3.1 form
Clear with micro- sem observation strychnia solubility micropin number, aciculiform is complete, and length is 384 μm, strychnia
Fig. 2 is shown in soluble micropin morphology investigation.
3.2 mechanical performance
It is punctured using aluminium foil and isolated rat skin penetrating tests to evaluate the puncture resistance of micropin.It, will when aluminium foil punctures
Micropin is placed horizontally on aluminium foil, applies the power of 10N to it, maintains the i.e. pierceable aluminium foil of 2min.The test of isolated rat skin penetrating
When, two pieces of rat skins are taken, micropin is lain against on one of skin, applies the power of 10N to it, rests on skin 3min,
Micropin is taken out, another piece of skin does not have to micropin and handles as a control group, and two pieces are dyed with methylene blue solution immediately, after 1min
Methylene blue solution is washed away with isopropanol reagent.
The result shows that: through micropin, treated that skin is difficult to clean after methylene blue staining, the skin without micropin processing
Methylene blue on skin can then be cleaned easily, show that the micropin can smoothly pierce through keratoderma, strychnia solubility is micro-
Fig. 3 is shown in pin puncture test.
The measurement of 4 drugloading rates
4.1 chromatographic condition
Chromatographic column: Agilent C18 (4.6mm × 250mm, 5um), mobile phase A (methanol)-Mobile phase B [water-acetic acid-three
Ethamine [230:2.4:0.3, (V/V/V)] (30:70, V/V)]；Detection wavelength is 265nm, flow velocity 1ml/min, 30 DEG C of column temperature, into
Sample amount is 10ul.
The preparation of 4.2 solution
4.2.1 the preparation of reference substance solution
Strychnia reference substance 0.00206g is weighed, it is accurately weighed, it is placed in 25ml volumetric flask, methanol is added to dissolve and determine
Hold, the strychnia reference substance solution that concentration is 80 μ g/ml is made.
4.2.2 the preparation of test solution
It is appropriate that precision weighs strychnia solubility micropin, is placed in 25ml volumetric flask, add proper amount of methanol ultrasound (power:
200w, frequency: 50kHz) processing 45min, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, after constant volume
Through the filtration of 0.22 μm of miillpore filter to get.
4.2.3 the preparation of blank micropin solution
It is appropriate that precision weighs blank micropin, obtains blank micropin solution by legal system below " 4.2.2 " item.
The test of 4.3 specificities
The reference substance solution under " 4.2 " item, strychnia solubility micropin solution are taken, each 10 μ l of blank micropin solution is pressed
Chromatographic condition sample introduction under " 4.1 " item shows that the method has specificity to strychnia.Strychnia reference substance in specificity test
HPLC chromatogram is shown in Fig. 4；Strychnia solubility micropin HPLC chromatogram is shown in Fig. 5 in specificity test；In specificity test
Strychnia feminine gender HPLC chromatogram is shown in Fig. 6.
The investigation of 4.4 linear relationships
Precision draws 0.5,2,4,6,8,10 μ l of strychnia reference substance solution under " 4.2 " item, by chromatostrip under " 4.1 " item
The measurement of part sample introduction does linear regression to drug sample volume (m) with peak area A, records chromatographic peak area.It is cross with sample volume (μ g)
Coordinate, peak area are that vertical sit carries out linear regression.Obtaining regression equation is Y=2E+06x+11263 (R2=0.9997), show horse
For money alkali in the 0.04 μ g range of μ g~0.8, peak area and reference substance sample volume are in good linear relationship.
3 strychnia reference substance standard curve of table
4.5 precision test
Precision draws strychnia reference substance solution 2.0ml, is settled to 5ml with mobile phase, and obtaining concentration is 0.032 μ g/ μ l
Strychnia reference substance solution, precision draw 10 μ l sample introductions, parallel 6 times, RSD% 1.41 shows that instrument precision is good.
4 Precision test result of table
4.6 repetitive test
It weighs with a collection of strychnia solubility micropin, is placed in 25ml volumetric flask, adds methanol to dissolve and be settled to scale,
By sample introduction under " 4.1 " item, parallel 6 parts, chromatogram is recorded, RSD 0.44% shows that method repeatability is good.
5 repetitive test result of table
4.7 stability test
Precision, which is drawn, to be measured with a strychnia test solution in 0,2,4,6,8,12,24,48h sample introduction, vomiting nut
The RSD of alkali peak area is 0.62%.
6 test solution stability test of table
The measurement of 4.8 rate of recovery
Appropriate strychnia solubility micropin powder is weighed, is added in the ratio that sample size and reference substance content are 1:1
Reference substance, is measured by chromatographic condition under " 4.1 " item by parallel 6 parts, calculate average recovery rate is 97.77%, RSD 2.75%,
Show that the reproducibility of the rate of recovery is good.
7 recovery test result of table
The measurement of 4.9 sample sizes
This product 3 batches are taken, according to strychnia content, every batch of are measured in parallel 3 times, survey in method measurement sample under content determination item
Surely 8 be the results are shown in Table.
8 strychnia solubility micropin sample size measurement result of table
The test of 5 strychnia solubility micropin Ligustrazine hydrochlorides
The processing of 5.1 rat skins
Healthy rat is taken, after anaesthetized with pentobarbital, shaving is carried out to rat abdomen with winged section's shaver immediately, at dislocation
Extremely, skin of abdomen is carefully removed, subcutaneous layer of fat is eliminated, is cleaned with physiological saline to no muddiness, is taken out, blotted with filter paper, it is standby
With.
The measurement of 5.2 Ligustrazine hydrochlorides
16 strychnia solubility micropin of Example, the power for applying 10N puncture skin and stick in skin with non-woven fabrics
On.Using vertical percutaneous dispersion test instrument, (effective diffusion area is 2.8cm2, receiving chamber volume is 7ml), mouse skin is placed in confession
To between pond and reception tank, cuticula is fixed tightly towards reception tank with clip towards supply pool, skin corium.With degassed processing
And the physiological saline of 20% ethyl alcohol of 37 DEG C of preheatings is receiving liquid, receiving chamber bath temperature is 37 DEG C, and magnetic stirring speed is
300r/min, as micropin experimental group；It separately takes a piece of micropin to be placed in supply pool, adds receiving liquid 1ml to dissolve it slowly, be used for
Comparative group, each parallel two parts, in 2h, 4h, 6h, 8h, 10h, 12h, for 24 hours, 36h, 48h sample 1ml, with 0.22 μm of miillpore filter mistake
Filter, high performance liquid chromatograph sample introduction measure the concentration of strychnia, and it is mutually synthermal to supply same volume immediately after every sub-sampling
Fresh receiving liquid calculates accumulation infiltration capacity.
Qn=(Cn×V0+∑Ci×V)/S
Q in formulanInfiltration capacity (ug/cm is accumulated for the unit area of n-th of sample point strychnia2),
CnFor the mass concentration (μ g/mL) of strychnia in n-th of sample point receiving liquid, V is reception tank volume (mL), V0
It is effective transmission area (cm when drug penetrates for sample volume (ml), S2), Ci is the i-th (connecing of measuring of i≤n-1 sample point
It receives liquid drug concentration (μ g/mL).
Table 9 accumulates infiltration capacity result table
As a result: the unit area transdermal penetration amount of micropin processing group is 7.9406 without micropin processing group in 48 hours
Times.
The drafting of 5.3 strychnia Ligustrazine hydrochloride curves
Using unit area accumulation infiltration capacity as ordinate, the time is abscissa, the strychnia transdermal penetration test of drafting
Curve is as shown in Figure 7.
The pharmacodynamic evaluation of 6 strychnia solubility micropins
6.1 experimental animal
SPF grades of SD rats (are provided) half male and half female, 180 ± 50g of weight by Chongqing Teng Xin Bioisystech Co., Ltd.
6.2 drug
Strychnia；Freund's complete adjuvant；
6.3 animal packet
Healthy rat is randomly divided into 16 groups: model control group, matrix group, strychnia solubility micropin administration group
(4 groups of embodiment, 6 groups of embodiment, 7 groups of embodiment, 8 groups of embodiment, 9 groups of embodiment, 10 groups of embodiment, is implemented 5 groups of embodiment
11 groups of example, 12 groups of embodiment, 13 groups of embodiment, 14 groups of embodiment, 15 groups of embodiment, 16 groups of embodiment), Normal group, often
6 healthy rats of group.
The preparation of 6.4 rheumatoid arthritis rat models
After remaining each group rat anesthesia outside Normal group, Yu Zuohou toes progress alcohol (75%) disinfection is simultaneously intradermal
Injection complete Freund's adjuvant (CFA) 0.1mL prepares rheumatoid arthritis inflammatory model.
6.5 administration
Start to be administered within second day after modeling success, in left back foot medicine-feeding, vomiting nut micropin administration group exists all formulations
Ankle gives a piece of strychnia solubility micropin (every micropin about 1.44mg containing strychnia), and matrix group is in identical bits
The blank micropin for giving a piece of not drug containing is set, is fixed after pressing 1min with medical proof fabric, model control group gives the life of same volume
Salt water 0.1mL is managed, Normal group is not processed, and successive administration 15 days.
The observation of 6.6 animal states and swelling degree of the paw measurement
Matrix group, model control group rat diet situation, mobility decrease after cause is scorching, and vola pedis and ankle-joint occur
Swelling, and it is rubescent, and increasingly aggravate；Rat diet situation, mobility are uninfluenced after the treatment of embodiment 4-16 group rat, foot
Plantar and ankle swelling, redness increasingly mitigate, and Normal group is without any exception.Each group rat is dry with strychnia micropin
The variation of degree of paw swelling is as shown in table 10 in prognosis 15 days.
Table 10: influence of the strychnia solubility micropin to degree of paw swelling
Note: compared with Normal group, P < 0.05 *；Compared with model control group, #P < 0.05.
6.7HE decoration method observes histotomy
Observe the feelings of each group synovial cells in rats, cartilage, inflammatory cell, pannus etc. under the microscope after HE is dyed
Condition.The synovium of joint epithelium marshalling of rats in normal control group as the result is shown has no pannus generation and inflammatory cell infiltration,
Boundary clear.The all visible synovial membrane epithelial proliferation of other each group rats, inflammatory cell infiltration are simultaneously generated with pannus, and cartilage is invaded
Erosion, mesostroma group, model control group rat articular gap are invisible, and strychnia solubility micropin group rabbit cartilage corrodes
Situation is lighter, the still visible joint space to narrow.
6.8Elisa method measures TNF-α, the content of IL-1 β in rat blood serum
Compared with Normal group, TNF-α, IL-1 β content significantly increase in model control group, bare substrate group serum, high
No significant difference (P > 0.05)；Compared with model control group, TNF-α, IL-1 β contain in embodiment 4-16 group rat blood serum
Amount significantly reduces, and difference has statistical significance (P < 0.05).As a result as shown in table 11.
Table 11: TNF-α, IL-1 β content results table in rat blood serum
Note: compared with model control group, P < 0.05 *
By pharmacodynamics test it is found that strychnia solubility micropin can be significant prepared by 4-16 of the embodiment of the present invention
Mitigate rat articular swelling, significantly reduce the level of TNF- ɑ and IL-1 β in serum, mitigates ankle-joint position synovial membrane epithelial proliferation
With cartilage erosion, joint space is maintained, there is good therapeutic effect to rat RA.
7 safety evaluatios
7.1 experimental animal
ICR mouse (is used for acute toxicity test), and SPF grades, weight 18-22g；SPF grades of SD rats (are tried for long term toxicity
Test and irritation test etc.), weight 180g or so.The equal half male and half female of rats and mice, by Chongqing, Teng Xin Bioisystech Co., Ltd is mentioned
For.
7.2 drugs and instrument
By strychnia solubility micropin prepared by embodiment 4-16；Operating scissors, eye scissors, tweezers, medicinal alcohol etc..
7.3 acute toxicity test
7.3.1 animal packet
Healthy mice is randomly divided into: (4 groups of embodiment is implemented for blank control group, strychnia solubility micropin administration group
5 groups of example, 6 groups of embodiment, 7 groups of embodiment, 8 groups of embodiment, 9 groups of embodiment, 10 groups of embodiment, 11 groups of embodiment, embodiment 12
Group, 13 groups of embodiment, 14 groups of embodiment, 15 groups of embodiment, 16 groups of embodiment), totally 16 groups, every group of 8 healthy mices.
7.3.2 administration
In addition to blank control group, strychnia solubility micropin administration group gives strychnia prepared by embodiment 4-16
Micropin processing (every microneedle array drugloading rate is 1.44mg or so), every rat gives two panels micropin, once a day.Rat body
Again based on 180g, is calculated according to the administration range 8-16mg/kg of 2.3.1 lower strychnias, give two panels micropin, that is, reach single
Maximum dosage-feeding.
7.3.3 method and result
Observe and record the toxic reaction and death condition on the same day after animal is administered.Including animal appearance, skin, behavior and
Reaction, breathing etc.；Continuous observation two weeks, after two weeks to still surviving animals carry out gross anatomy, observation the heart, liver, spleen, lung, kidney and
The change of the main organs such as stomach and intestine and tissue.
Blank group is without any exception；It is dead without animal in the strychnia solubility micropin processing group mouse administration same day to two weeks
It dies, exception is showed no to extraneous reaction, appearance, behavior and breathing, glandular secretion etc., does not observe obvious poisoning symptom, mouse
The internal organs no abnormality seen such as thoracic cavity, the heart, liver, spleen, lung, kidney and stomach and intestine.
The irritation test of 7.4 pairs of skin
Healthy rat is taken, is anaesthetized, depilation processing is carried out to administration area back.Administration before check skin of unhairing whether because
Unhairing and be damaged, select the good rat of skin condition it is spare.It is given along dorsal midline selection left or right side position a piece of
Strychnia solubility microneedle patch (drugloading rate 1.44mg), and further fixed with medical proof fabric.Micropin administration 1h, 12h,
For 24 hours, visually observe whether medicine-feeding part erythema and edema occurs after 36h.As a result, it has been found that not pierced when administration 1h, 12h to skin
Swash property；Slightly there is red dot, no edema at micropin piercing when being administered for 24 hours；Red dot almost disappears when 36h is administered.It is believed that
Prepared strychnia solubility micropin is almost nonirritant.
7.5 strychnia solubility micropin long-term dosing studies
Healthy rat is taken, after being anaesthetized, removes the hair at knee rat articular position, fixed rat.With thumb by one
Piece strychnia solubility microneedle patch (drugloading rate 1.44mg) presses on the articular skin position of hair removal, stops 1min
Afterwards, strychnia solubility micropin is fixed with medical proof fabric, strychnia solubility micropin, administration one in one day is taken out after 30min
Secondary, preceding and medicine-feeding part after administration 15 days skin conditions are administered in observation.Observe whether rat diet activity etc. has exception simultaneously,
And to strychnia solubility micropin administration group rat carry out gross anatomy, observe long term administration after its heart, liver, spleen, lung, kidney,
Whether stomach, intestines etc. have exception.After strychnia solubility micropin is administered 15 days, rat knee joints area skin has slight damage,
Skin can be damaged but lesser extent by showing the administration for a long time of strychnia solubility micropin；Rat does not go out in administration 15 days
Existing death, the Non Apparent Abnormalities such as diet, behavior, the Non Apparent Abnormalities such as its its heart, liver, spleen, lung, kidney, stomach, intestines after dissection, therefore institute
The safety of the strychnia solubility micropin of preparation is higher.
8 summarize
(1) present invention is host material using polyvinylpyrrolidone and chondroitin sulfate, takes vacuum decompression method, is made
Standby strychnia solubility micropin morphology and satisfactory mechanical property.
(2) strychnia is prepared into biodegradable micropin, not only acts as slow releasing function, patient need not repeatedly dressing,
The compliance for improving patient, solves the problems, such as half-life short, moreover it is possible to penetrate cuticula, drug release is promoted to play drug effect, solution
Drug of having determined is difficult to the problem through cuticula.
(3) strychnia content and transdermal test in vitro effect in high effective liquid chromatography for measuring sample are used, strychnia
Equation of linear regression is Y=2E+06x+11263 (R2=0.9997), the range of linearity is 0.04 μ of μ g~0.8 g；Strychnia can
The unit area transdermal penetration amount of micropin processing group is 7.9406 times without micropin processing group in dissolubility micropin 48 hours.
(4) the strychnia content assaying method that the present invention establishes is simply accurate, and Feasible degree is high.
(5) present invention by test has searched out the material that matter is soft, nonirritant, due to take vacuum decompression method and
Take one block of non-woven fabrics to be directly laid in the stromal surface after depressurizing after decompression, the dry sequence of matrix be by pin hole slowly to
Up to back sheet, micropin prepared by the present invention has reached the soft form of needle dura mater, makes patient to can move freely since the medicine.
(6) preparation process of strychnia solubility micropin and supplementary material are screened by stringent science, significant in efficacy,
It has no toxic side effect, solves strychnia effective dose and toxic dose ten in terms for the treatment of rheumatoid arthritis disease and tap
Closely, the problem of seriously limiting its application clinically.
(7) strychnia solubility micropin prepared by the present invention can significantly mitigate rat articular swelling, significantly reduce blood
The level of TNF- ɑ and IL-1 β in clear, mitigate ankle-joint position synovial membrane epithelial proliferation and cartilage erosion, joint space are maintained, to big
Mouse RA has good therapeutic effect.
(8) present invention is proved by safety testing, and safe without toxic side effect is used in dosage range.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
State, but on the basis of the present invention, it can be made it is some modify or improve, this is aobvious and easy to those skilled in the art
See.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of strychnia solubility micropin, it is characterised in that be made according to the following ratio of following component: strychnia
0.012002 part~0.012010 part, 0.998 part~1.006 parts of chondroitin sulfate, 0.997 part of polyvinylpyrrolidone~
1.005 part.
2. a kind of strychnia solubility micropin according to claim 1, it is characterised in that by following component by matching below
Than being made: 0.012004 part~0.012008 part of strychnia, 1.000 parts~1.004 parts of chondroitin sulfate, polyvinylpyrrolidine
0.999 part~1.003 parts of ketone.
3. a kind of strychnia solubility micropin according to claim 1, it is characterised in that by following component by matching below
Than being made: 0.012006 part of strychnia, 1.002 parts of chondroitin sulfate, 1.001 parts of polyvinylpyrrolidone.
4. a kind of method for preparing any one of claim 1-3 strychnia solubility micropin, it is characterised in that specific step
Suddenly are as follows:
1) strychnia of formula ratio is weighed, 1.4ml~1.8ml pure water is added, ultrasound makes it dissolve, and obtains strychnia drug
Aqueous solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia pharmaceutical aqueous solution is added after mixing in chondroitin sulfate
In, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 2h~4h, is put into 18 DEG C~22 DEG C vacuum ovens, is depressurized；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 3 days~5 days, demoulding, obtain strychnia solubility
Micropin.
5. preparing the method for strychnia solubility micropin according to claim 4, it is characterised in that specific steps are as follows:
1) strychnia of formula ratio is weighed, 1.6ml pure water is added, ultrasound makes it dissolve, and obtains strychnia pharmaceutical aqueous solution；
2) polyvinylpyrrolidone of formula ratio is weighed, step 1) strychnia pharmaceutical aqueous solution is added after mixing in chondroitin sulfate
In, quickly stirring makes to form uniform solution；
3) it is poured into micropin mold after being swollen 3h, is put into 20 DEG C of vacuum ovens, is depressurized；
4) it takes out to stand overnight defoaming in interior and move back into drier and solidifies 4 days, demoulding, obtain strychnia solubility micropin.
6. according to the preparation method of any one of claim 4 or 5 the strychnia solubility micropin, it is characterised in that described to subtract
Press strip part is 0.07MPa~0.09MPa.
7. according to the preparation method of any one of claim 4 or 5 the strychnia solubility micropin, it is characterised in that described to subtract
The pressure time is 25min~35min.
8. a kind of method of any one of measurement claim 1-3 strychnia solubility micropin, it is characterised in that according to efficient
Liquid chromatography for measuring, specific measuring method are as follows:
(1) chromatographic condition: Agilent C18 4.6mm × 250mm, 5um, mobile phase A: Mobile phase B is 30:70 by volume,
Mobile phase A is methanol, and Mobile phase B is water 230 by volume: acetic acid 2.4: triethylamine 0.3, Detection wavelength 265nm, flow velocity
1ml/min, 30 DEG C of column temperature；
(2) preparation of solution
1) preparation of reference substance solution
Strychnia reference substance 0.00206g is weighed, it is accurately weighed, it is placed in 25ml volumetric flask, adds methanol dissolution and constant volume, system
Obtain the strychnia reference substance solution that concentration is 80 μ g/ml；
2) preparation of test solution
Precision weighs strychnia solubility micropin 0.1g, is placed in 25ml volumetric flask, adds 25ml methanol in power 200w, frequency
45min~50min is ultrasonically treated under 50kHz, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, constant volume
By the filtration of 0.22 μm of miillpore filter to get；
3) preparation of blank micropin solution
Precision weighs blank micropin 0.1g, is placed in 25ml volumetric flask, and 25ml methanol is added to surpass at power 200w, frequency 50kHz
Sonication 45min~50min, the structure for destroying micropin makes to contain sufficiently to be released in drug therein, through 0.22 μ after constant volume
M miillpore filter filters to get blank micropin solution；
(3) it measures
1) specificity measures
Take the reference substance solution under Section 2, test solution, each 10 μ l of blank micropin solution, by chromatostrip under Section 1
Part sample introduction, measurement；
2) linear relationship measures
Precision draws strychnia reference substance solution 0.5 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l, by Section 1 under Section 2
Lower chromatographic condition sample introduction, measurement；
3) precision measures
Precision draws strychnia reference substance solution 2.0ml under Section 2, is settled to 5ml with mobile phase, and obtaining concentration is 0.032
The strychnia reference substance solution of μ g/ μ l, precision draw 10 μ l sample introductions, by chromatographic condition sample introduction under Section 1, measurement；
4) repeatability measurement
It weighs with a collection of strychnia solubility micropin, is placed in 25ml volumetric flask, adds methanol to dissolve and be settled to scale, it is accurate
10 μ l are drawn, by chromatographic condition sample introduction under Section 1, measurement；
5) Stability Determination
Precision is drawn with a 10 μ l of strychnia solubility test solution in 0h, 2h, 4h, 6h, 8h, 12h, and for 24 hours, 48h is pressed
Chromatographic condition sample introduction under Section 1, measurement；
6) measurement of the rate of recovery
Appropriate strychnia solubility micropin powder is weighed, is added and compares in the ratio that sample size and reference substance content are 1:1
Product, precision are drawn 10 μ l and are measured by chromatographic condition under Section 1；
7) sample size measures
Take the reference substance solution under Section 2, test solution, each 10 μ l of blank micropin solution, by chromatostrip under Section 1
Part sample introduction measures strychnia content in sample.
9. strychnia solubility micropin measuring method according to claim 8, it is characterised in that the blank micropin
Preparation method is the following steps are included: weigh polyvinylpyrrolidone 0.500g, chondroitin sulfate 0.500g, be added 0.7ml~
0.9ml pure water is stirred evenly rapidly with glass bar, is cast on micropin mold after being swollen 2h~4h, is placed in 18 DEG C~22 DEG C
Vacuum oven in, be evacuated to 0.05MPa~0.09MPa, depressurize 25min~35min, taking-up is placed in 2 DEG C~6 DEG C refrigerators
It is interior, stand overnight defoaming move back to drier solidify 3~5 days, demoulding to get.
10. a kind of strychnia solubility micropin, it is characterised in that the strychnia solubility micropin is in treatment rheumatoid
Application in terms of arthritis disease.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810597A (en) * | 2010-04-26 | 2010-08-25 | 南京中医药大学 | Transdermal patch containing vauqueline and preparation method and application thereof |
| CN106039552A (en) * | 2016-05-30 | 2016-10-26 | 北京化工大学 | Bubble-type microneedle and preparation method therefor |
| WO2017018086A1 (en) * | 2015-07-29 | 2017-02-02 | 日本写真印刷株式会社 | Microneedle sheet |
| WO2017043627A1 (en) * | 2015-09-11 | 2017-03-16 | 株式会社バイオセレンタック | Microneedle preparation |
| CN106619480A (en) * | 2016-10-20 | 2017-05-10 | 南通普莱德医疗器械科技有限公司 | Novel polymer micro-needle array and preparation method thereof |
| US20180185625A1 (en) * | 2017-01-05 | 2018-07-05 | Quadmedicine | Method of manufacturing microneedle and microneedle manufactured thereby |
| CN108498936A (en) * | 2017-02-28 | 2018-09-07 | 富士胶片株式会社 | The manufacturing method of sheet material with needle-shaped protrusion |
| CN108524916A (en) * | 2018-05-31 | 2018-09-14 | 浙江中医药大学 | A kind of preparation method for the soluble micropin carrying Neurotoxin From Naja Naja Atra |
| CN108939280A (en) * | 2018-04-13 | 2018-12-07 | 杭州电子科技大学 | A kind of preparation method of SU8 microneedle array patch |
| CN109200012A (en) * | 2017-07-06 | 2019-01-15 | 中科微针（北京）科技有限公司 | A kind of layering dissolution micropin containing inorganic salts |
-
2019
- 2019-02-23 CN CN201910134480.3A patent/CN109884223A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810597A (en) * | 2010-04-26 | 2010-08-25 | 南京中医药大学 | Transdermal patch containing vauqueline and preparation method and application thereof |
| WO2017018086A1 (en) * | 2015-07-29 | 2017-02-02 | 日本写真印刷株式会社 | Microneedle sheet |
| WO2017043627A1 (en) * | 2015-09-11 | 2017-03-16 | 株式会社バイオセレンタック | Microneedle preparation |
| CN106039552A (en) * | 2016-05-30 | 2016-10-26 | 北京化工大学 | Bubble-type microneedle and preparation method therefor |
| CN106619480A (en) * | 2016-10-20 | 2017-05-10 | 南通普莱德医疗器械科技有限公司 | Novel polymer micro-needle array and preparation method thereof |
| US20180185625A1 (en) * | 2017-01-05 | 2018-07-05 | Quadmedicine | Method of manufacturing microneedle and microneedle manufactured thereby |
| CN108498936A (en) * | 2017-02-28 | 2018-09-07 | 富士胶片株式会社 | The manufacturing method of sheet material with needle-shaped protrusion |
| CN109200012A (en) * | 2017-07-06 | 2019-01-15 | 中科微针（北京）科技有限公司 | A kind of layering dissolution micropin containing inorganic salts |
| CN108939280A (en) * | 2018-04-13 | 2018-12-07 | 杭州电子科技大学 | A kind of preparation method of SU8 microneedle array patch |
| CN108524916A (en) * | 2018-05-31 | 2018-09-14 | 浙江中医药大学 | A kind of preparation method for the soluble micropin carrying Neurotoxin From Naja Naja Atra |
Non-Patent Citations (8)
| Title |
|---|
| KAI-JEN HSUEH 等: "Transcutaneous immunization of Streptococcus suis bacterin using dissolving microneedles", 《COMPARATIVE IMMUNOLOGY, MICROBIOLOGY》 * |
| 何晓玮 等: "马钱子碱新型给药系统治疗类风湿性关节炎的研究进展", 《中华中医药学刊》 * |
| 刘斌 等: "《中药有效部位及成分提取工艺和检测方法》", 31 July 2007, 中国中医药出版社 * |
| 方亮 等: "《《药剂学》应试指南与习题解析》", 30 September 2011, 中国医药科技出版社 * |
| 杜冠峰 等: "中药镇痛活性成分经皮给药研究进展", 《安徽医药》 * |
| 王艳宏 等: "不同载药形式的肉桂油对马钱子总碱中马钱子碱和士的宁体外透皮吸收的影响", 《中国药房》 * |
| 管庆霞 等: "马钱子碱及其纳米结构脂质载体在大鼠体内的药动学比较研究", 《中国药房》 * |
| 轻工业标准化编辑出版委员会: "《中国轻工业标准汇编 化妆品卷 第2版》", 31 May 2012, 中国轻工业出版社 * |
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