CN102988614A - Preparation method and application of antiphlogosis tablet - Google Patents
Preparation method and application of antiphlogosis tablet Download PDFInfo
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- CN102988614A CN102988614A CN2012103784346A CN201210378434A CN102988614A CN 102988614 A CN102988614 A CN 102988614A CN 2012103784346 A CN2012103784346 A CN 2012103784346A CN 201210378434 A CN201210378434 A CN 201210378434A CN 102988614 A CN102988614 A CN 102988614A
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- 238000000194 supercritical-fluid extraction Methods 0.000 claims abstract description 10
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 claims abstract description 5
- 208000033826 Promyelocytic Acute Leukemia Diseases 0.000 claims abstract description 5
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- FXNFHKRTJBSTCS-UHFFFAOYSA-N Baicalein Natural products C=1C(=O)C=2C(O)=C(O)C(O)=CC=2OC=1C1=CC=CC=C1 FXNFHKRTJBSTCS-UHFFFAOYSA-N 0.000 abstract 1
- 244000035851 Chrysanthemum leucanthemum Species 0.000 abstract 1
- 235000008495 Chrysanthemum leucanthemum Nutrition 0.000 abstract 1
- 240000001949 Taraxacum officinale Species 0.000 abstract 1
- 235000005187 Taraxacum officinale ssp. officinale Nutrition 0.000 abstract 1
- 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
- 229940015301 baicalein Drugs 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 230000000452 restraining effect Effects 0.000 abstract 1
- 229960004756 ethanol Drugs 0.000 description 21
- IPQKDIRUZHOIOM-UHFFFAOYSA-N Oroxin A Natural products OC1C(O)C(O)C(CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IPQKDIRUZHOIOM-UHFFFAOYSA-N 0.000 description 10
- 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
- 229960003321 baicalin Drugs 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
- 230000010261 cell growth Effects 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
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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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- JWHHANVGNNWIRI-UHFFFAOYSA-N methanol phosphoric acid hydrate Chemical compound O.OC.OP(O)(O)=O JWHHANVGNNWIRI-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 the 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, is made as crude drug by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, can anti-inflammation.Be used for respiratory tract infection, heating, pneumonia, bronchitis brings up phlegm when one coughs, furuncle and phyma etc.
In the prior art, not yet there is Pianomide to adopt the report of supercritical and microwave technology aspect the preparation extracting, and adopts 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 used 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 the application of a kind of Pianomide in preparation inhibition people acute promyelocytic leukemia cell NB4 cell proliferation medicine.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of preparation method of Pianomide, made as crude drug by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, described method is comprised of the following step: get Flos Chrysanthemi Indici, join in the 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 gets supercritical extract, and is for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in the 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 the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
The preparation method of above-mentioned a kind of Pianomide, described CO
2The percent by volume that the 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.
The application of above-mentioned Pianomide in preparation inhibition people acute promyelocytic leukemia cell NB4 cell proliferation medicine.
In the 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 the 2-3 sheet at every turn, takes 3-4 time in 1st, 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 the Pianomide of test one, distinct methods preparation
1, instrument and reagent Pianomide of the present invention: press the preparation of embodiment 3 methods, use the 1784g crude drug, make 500 through extraction, every heavy 0.5g.Former Pianomide by the preparation of ministry standard method, uses the 1784g crude drug, makes 500 through extraction, 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: be filler with octadecylsilane chemically bonded silica; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; The detection wavelength is 280nm.Number of theoretical plate is pressed the baicalin peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours baicalin reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methanol and makes the solution that every 1ml contains 18 μ g, and get final product.
The preparation of need testing solution: get Pianomide of the present invention and former Pianomide, porphyrize, mixing is got 1g, and is accurately weighed, the accurate 70% ethanol 20ml that adds, close plug, supersound process 10 minutes, centrifugal, get supernatant, and get final product.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product.
3, result
The result shows that the content of baicalin is the 1.64mg/ sheet in the Pianomide of the present invention; And the content of baicalin is the 0.28mg/ sheet in the former Pianomide, and in the situation that dose reduces, baicalin content improves a lot.
Above-mentioned studies show that, the Pianomide that adopts the present invention to prepare, 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 that the scope of the above-mentioned theme of the present invention only limits to following example, all technology that realizes 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, with Flos Chrysanthemi Indici, join in the 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, get supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 400W extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of baicalin is the 1.59mg/ sheet in the finished product.
Embodiment 2
Get Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, with Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 600W extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of baicalin is the 1.57mg/ sheet in the finished product.
Embodiment 3
Get Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, with Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 500W extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content of baicalin is the 1.64mg/ sheet in the finished product.
Embodiment 4: Pianomide suppresses the experimentation data of NB4 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing the 100mg Pianomide, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ l doff manage packing ,-20 ℃ of storages, and 0.2 μ m filter filters dehydrated alcohol in order to the usefulness of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061 Lot.No.758137 of DMEM(GIBCO company); Hyclone (Hangzhoupro, sky, Zhejiang bio tech ltd Lot.No.100419); NaHCO3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033 Lot.No.1088387 of a specified duration); 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); As seen-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 makes 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 (the accurate experimental facilities in Shanghai 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) the NB4 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells during to logarithmic (log) phase, collecting cell discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, behind 37 ℃ of digestion 2min, to wherein adding 5ml complete medium neutralization reaction, behind the piping and druming cell it is changed in the centrifuge tube, the centrifugal 5min of 1000rpm adjusts 3 * 104/ml of concentration of cell suspension.
2) the 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 the Growth of Cells situation, generally grow to 50%-70%, add Pianomide solution, continue to cultivate 24h.
4) add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) behind the 24h, continue to cultivate 4h.
5) the buckle method is removed supernatant behind the 4h, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on the shaking table, and crystal is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 490nm place.
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) is set 6 multiple holes for every group.
7) result represents with the suppression ratio of medicine to cell: 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 the mtt assay experiment, compare with matched group, 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), and utmost point significant difference (P＜0.001) is arranged when dosage reaches 15-20mg/ml.
Table 1 Pianomide is on NB4 cell inhibitory effect impact research
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
Pianomide can suppress the NB4 cell proliferation, reduces the Growth of Cells number of NB4 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of a Pianomide is made as crude drug by Herba Taraxaci 446g, Herba Violae 446g, Flos Chrysanthemi Indici 446g, Radix Scutellariae 446g, it is characterized in that described method is comprised of the following step: get Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 2L, drop in the 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 the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting is made 500, every heavy 0.5g.
2. the preparation method of described a kind of Pianomide according to claim 1 is characterized in that described CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
3. the preparation method of described a kind of Pianomide according to claim 1 is characterized in that described microwave extracting power 500W, extracts 6 minutes at every turn.
4. the preparation method of described a kind of Pianomide according to claim 1 is characterized in that described CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
5. the according to claim 1 application of described a kind of Pianomide in preparation inhibition people acute promyelocytic leukemia cell NB4 cell proliferation medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103566023A (en) * | 2013-10-29 | 2014-02-12 | 山东省立医院 | Preparation method and application of toothache anti-inflammation tablets |
| CN105168624A (en) * | 2015-08-27 | 2015-12-23 | 韩志强 | Anti-inflammatory tablet and preparation method thereof |
| CN105911161A (en) * | 2016-04-12 | 2016-08-31 | 吉林修正药业新药开发有限公司 | Anti-inflammatory tablet HPLC fingerprint construction method |
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| CN1733079A (en) * | 2005-09-05 | 2006-02-15 | 石洪波 | Pharmaceutical composition for treating and preventing respiratory tract viral infection, its preparation process and application |
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| CN1733079A (en) * | 2005-09-05 | 2006-02-15 | 石洪波 | Pharmaceutical composition for treating and preventing respiratory tract viral infection, its preparation process and application |
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| CN103566023B (en) * | 2013-10-29 | 2015-11-18 | 山东省立医院 | A kind of preparation method of toothache Pianomide and application |
| CN105168624A (en) * | 2015-08-27 | 2015-12-23 | 韩志强 | Anti-inflammatory tablet and preparation method thereof |
| CN105168624B (en) * | 2015-08-27 | 2019-11-29 | 韩志强 | A kind of sulfathiazole and preparation method thereof |
| CN105911161A (en) * | 2016-04-12 | 2016-08-31 | 吉林修正药业新药开发有限公司 | Anti-inflammatory tablet HPLC fingerprint construction method |
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