CN102988614B - Preparation method and application of antiphlogosis tablet - Google Patents
Preparation method and application of antiphlogosis tablet Download PDFInfo
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- 244000035851 Chrysanthemum leucanthemum Species 0.000 abstract 1
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- 240000001949 Taraxacum officinale Species 0.000 abstract 1
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- UDFLTIRFTXWNJO-UHFFFAOYSA-N baicalein Chemical compound O1C2=CC(=O)C(O)=C(O)C2=C(O)C=C1C1=CC=CC=C1 UDFLTIRFTXWNJO-UHFFFAOYSA-N 0.000 abstract 1
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- IKIIZLYTISPENI-ZFORQUDYSA-N baicalin Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IKIIZLYTISPENI-ZFORQUDYSA-N 0.000 description 10
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- 239000013558 reference substance Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a preparation method of an antiphlogosis tablet which is prepared from crude drugs including 446g of dandelion, 446g of herba violae, 446g of wild chrysanthemum and 446g of radix scutellariae through supercritical extraction and microwave extraction, so that the content of baicalein is greatly improved. The invention also provides the application of the antiphlogosis tablet in preparing a medicine for restraining proliferation of human acute promyelocytic leukemia cell, NB4 cell.
Description
Technical field
The present invention relates to Chinese medicine preparation technical field, be specifically related to a kind of preparation method and application of Pianomide.
Background technology
Pianomide is recorded in Ministry of Public Health standard WS3-B-0809-91, by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, as crude drug, is made, can anti-inflammation.For respiratory tract infection, heating, pneumonia, bronchitis, brings up phlegm when one coughs, furuncle and phyma etc.
In prior art, not yet there is Pianomide to adopt the report of supercritical and microwave technology extracting aspect preparation, and adopt the method that powder and decocting boil of beating, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
Goal of the invention: in order to address the above problem, the object of the present invention is to provide a kind of preparation method of Pianomide.
Another object of the present invention is to provide a kind of Pianomide to suppress the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of Pianomide, by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, as crude drug, made, described method is comprised of the following step: get Flos Chrysanthemi Indici, join in CO2 supercritical extraction device, ethanol is as entrainer, the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO2 flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400-600W, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
The preparation method of above-mentioned a kind of Pianomide, described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
The preparation method of above-mentioned a kind of Pianomide, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of Pianomide, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned Pianomide suppresses the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation.
In prior art, every 0.5g of Pianomide, each 4-6 sheet, 3-4 time on the one, every 0.5g of Pianomide that employing the present invention is prepared into only needs 2-3 sheet at every turn, within 1st, takes 3-4 time, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of baicalin content in Pianomide prepared by test one, distinct methods
1, instrument and reagent Pianomide of the present invention: press embodiment 3 method preparations, use 1784g crude drug, make 500 through extracting, every heavy 0.5g.Former Pianomide, by the preparation of ministry standard method, is used 1784g crude drug, through extracting, makes 500, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLER AE240 electronic analytical balance; Baicalin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica, be filler; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; Detection wavelength is 280nm.Number of theoretical plate is pressed baicalin peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes at 60 ℃ of drying under reduced pressure baicalin reference substance of 4 hours appropriate, adds methanol and makes every 1ml containing the solution of 18 μ g, obtains.
The preparation of need testing solution: get Pianomide of the present invention and former Pianomide, porphyrize, mixes, and gets 1g, accurately weighed, precision adds 70% ethanol 20ml, close plug, supersound process 10 minutes, centrifugal, get supernatant, obtain.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
3, result
Result shows, in Pianomide of the present invention, the content of baicalin is 1.64mg/ sheet; And the content of baicalin is 0.28mg/ sheet in former Pianomide, in the situation that dose reduces, baicalin content improves a lot.
Above-mentioned research shows, the Pianomide that adopts the present invention to prepare, and active constituent content is higher than the standby Pianomide of ministry standard legal system.
The specific embodiment
Form by the following examples, foregoing of the present invention is described in further detail again, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, by Flos Chrysanthemi Indici, join in CO2 supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, 30 ℃ of temperature, CO2 flow 1m1/g crude drug min, extraction time 150min, obtain supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400W, extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is 1.59mg/ sheet.
Embodiment 2
Get Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, by Flos Chrysanthemi Indici, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 30MPa, temperature 50 C, CO
2flow 3m1/g crude drug min, extraction time 180min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is 1.57mg/ sheet.
Embodiment 3
Get Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, by Flos Chrysanthemi Indici, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 20MPa, 40 ℃ of temperature, CO
2flow 2m1/g crude drug min, extraction time 160min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 500W, extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is 1.64mg/ sheet.
Embodiment 4: Pianomide suppresses the experimentation data of NB4 cell proliferation
1 experiment material
1.1 experiment cell strains
People's acute promyelocytic leukemia cell NB4 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Pianomide of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg Pianomide, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe subpackages ,-20 ℃ of storages, 0.2 μ m filter filters dehydrated alcohol in order to the use of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061 Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The Cat.No.11810-033 Lot.No.1088387 of NaHCO3(Shanghai Jiu Yi chemical reagent company limited); Trypsin(AMRESCO company lot number: 2010/04); EDTA(AMRESCO company lot number: 2009/10); Penicillin G Sodium Salt(AMRESCO company lot number: 2010242); Streptomycin Sulfate(AMRESCO company lot number: 2010382); Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182); MTT (Biosharp lot number: 0793); The autogamy of PBS(laboratory);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX 190); CO2 incubator (FORMA model: 3111); (safe and sound company of Su Jing group manufactures model to super-clean bench: SW-CJ-ZFD); Pure water instrument (U.S. Spring company model: S/N 020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (Shanghai accurate experimental facilities company model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000); 0.2 μ m filter (MILLIPORE model: SLGP033RB); 10cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2 experimental techniques
1) NB4 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells is during to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, after 37 ℃ of digestion 2min, add wherein 5ml complete medium neutralization reaction, after piping and druming cell, proceeded in centrifuge tube, the centrifugal 5min of 1000rpm, adjusts 3 * 104/ml of concentration of cell suspension.
2) cell kind is entered in 96 well culture plates, every hole adds cell suspension 180 μ l, culture plate put into cell culture incubator (37 ℃, 5%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add Pianomide solution, continue to cultivate 24h.
4) after 24h, add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT), continue to cultivate 4h.
5) after 4h, buckle method is removed supernatant, with absorbent paper, pats dry gently, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, and crystal is fully dissolved.At enzyme-linked immunosorbent assay instrument 490nm place, measure the light absorption value in each hole.
6) background (do not add cell, only add culture fluid) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide), sets 6 multiple holes for every group.
7) result represents the suppression ratio of cell with medicine: cell increment suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value * 100%.Experiment repeats 3 times.
3 statistical dispositions
Adopt correlation analysis and Student t check in Microsoft Excel 2003 softwares, data represent with mean ± S.D..
4 experimental results
Statistical result showed after mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to NB4 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has utmost point significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 Pianomide is on NB4 cell inhibitory effect impact research
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Pianomide can suppress NB4 cell proliferation, reduces the Growth of Cells number of NB4 cell, and this effect is dose dependent.
Claims (4)
1. a Pianomide suppresses the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation, it is characterized in that Pianomide made as crude drug by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, preparation method is comprised of the following step: get Flos Chrysanthemi Indici, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO
2flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400-600W, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 500, every heavy 0.5g.
2. a kind of Pianomide suppresses the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. a kind of Pianomide suppresses the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation according to claim 1, it is characterized in that the power of microwave extracting described in preparation method 500W, extracts 6 minutes at every turn.
4. a kind of Pianomide suppresses the application in people's acute promyelocytic leukemia cell NB4 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
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| CN105168624B (en) * | 2015-08-27 | 2019-11-29 | 韩志强 | A kind of sulfathiazole and preparation method thereof |
| CN105911161B (en) * | 2016-04-12 | 2018-03-27 | 吉林修正药业新药开发有限公司 | A kind of sulfathiazole HPLC fingerprint map construction methods |
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