CN102988479A - Preparation method and application of anti-dazzling tablet - Google Patents
Preparation method and application of anti-dazzling tablet Download PDFInfo
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- 208000032839 leukemia Diseases 0.000 claims abstract description 5
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- 230000005764 inhibitory process Effects 0.000 claims description 3
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 abstract description 9
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 abstract description 9
- 229940114124 ferulic acid Drugs 0.000 abstract description 9
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 abstract description 9
- 235000001785 ferulic acid Nutrition 0.000 abstract description 9
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 abstract description 9
- 241000112528 Ligusticum striatum Species 0.000 abstract 1
- 235000006679 Mentha X verticillata Nutrition 0.000 abstract 1
- 235000002899 Mentha suaveolens Nutrition 0.000 abstract 1
- 235000001636 Mentha x rotundifolia Nutrition 0.000 abstract 1
- 229910052602 gypsum Inorganic materials 0.000 abstract 1
- 239000010440 gypsum Substances 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 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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- 239000012531 culture fluid Substances 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 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
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 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
- 238000002965 ELISA Methods 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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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a preparation method of an anti-dazzling tablet which is prepared from crude drugs including 200g of Ligusticum wallichii, 200g of radix angelicae, 100g of mint, 100g of schizonepetaspike and 100g of gypsum through supercritical extraction and microwave extraction, so that the content of ferulic acid is greatly improved. The invention also relates to the application of the anti-dazzling tablet in preparing a medicine for restraining proliferation of human leukemia cell, K562 cell.
Description
Technical field
The present invention relates to the Chinese medicine preparation technical field, be specifically related to a kind of clear dizzy preparation method and application.
Background technology
Clear dizzy is recorded in Ministry of Public Health standard WS3-B-0836-91, is made as crude drug by Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, can dispeling wind and relieving fever.Be used for wind heat and have a dizzy spell, migraine and general headache, nasal obstruction toothache.
In the prior art, not yet there be dizzy clearly to adopt the report of supercritical and microwave technology aspect the preparation extracting, 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 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 clear dizzy preparation method.
Another object of the present invention is to provide a kind of clear dizzy application in preparation inhibition Leukemia K562 cell cell proliferation medicine.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of clear dizzy preparation method, made as crude drug by Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, described method is comprised of the following step: get Rhizoma Chuanxiong, Herba Menthae, Herba Schizonepetae, 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 1-3ml/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.
Above-mentioned a kind of clear dizzy preparation method, described CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
Above-mentioned a kind of clear dizzy preparation method, described microwave extracting power 500W extracts 6 minutes at every turn.
Above-mentioned a kind of clear dizzy preparation method, described CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned clear dizzy application in preparation inhibition Leukemia K562 cell cell proliferation medicine.
In the prior art, clear dizzy every 0.5g, each 4,2 times on the one, the clear dizzy every 0.5g that adopts the present invention to be prepared into, 2 of need were only taken 2 times on the 1st at every turn, had greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of clear dizzy middle ferulaic acid content of test one, distinct methods preparation
1, instrument and reagent the present invention are clear dizzy: press the preparation of embodiment 3 methods, use the 700g crude drug, make 500 through extraction, every heavy 0.5g.Former clear dizzy, by the preparation of ministry standard method, use the 700g crude drug, make 500 through extraction, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLER AE240 electronic analytical balance; Ferulic acid 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 ferulic acid peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours ferulic acid 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 clear dizzy and former clear dizzy of the present invention, 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 the clear dizzy middle ferulic acid of the present invention is the 1.26mg/ sheet; And the content of former clear dizzy middle ferulic acid is the 0.25mg/ sheet, and in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned studies show that, clear dizzy of adopting the present invention's preparation, active constituent content is higher than standby clear dizzy 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 Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, with Rhizoma Chuanxiong, Herba Menthae, Herba Schizonepetae, 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 500, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.29mg/ sheet in the finished product.
Embodiment 2
Get Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, with Rhizoma Chuanxiong, Herba Menthae, Herba Schizonepetae, 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 500, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.32mg/ sheet in the finished product.
Embodiment 3
Get Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, with Rhizoma Chuanxiong, Herba Menthae, Herba Schizonepetae, 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 500, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.26mg/ sheet in the finished product.
Embodiment 4: clear dizzy experimentation data that suppresses the K562 cell proliferation
1 experiment material
1.1 experiment cell strain
The Leukemia K562 cell cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: clear dizzy of the present invention: press the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing clear dizzy of 100mg, 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.11810-033Lot.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 K562 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 clear dizzy 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 K562 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 clear dizzy on K562 cell inhibitory effect impact research
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
Clear dizzy can be suppressed the K562 cell proliferation, reduces the Growth of Cells number of K562 cell, and this effect is dose dependent.
Claims (5)
1. a clear preparation method of dizzy is made as crude drug by Rhizoma Chuanxiong 200g, Radix Angelicae Dahuricae 200g, Herba Menthae 100g, Herba Schizonepetae 100g, Gypsum Fibrosum 100g, it is characterized in that described method is comprised of the following step: get Rhizoma Chuanxiong, Herba Menthae, Herba Schizonepetae, 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. described a kind of clear dizzy preparation method 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. described a kind of clear dizzy preparation method according to claim 1 is characterized in that described microwave extracting power 500W, extracts 6 minutes at every turn.
4. described a kind of clear dizzy preparation method 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. according to claim 1 described a kind of clear dizzy application in preparation inhibition Leukemia K562 cell cell proliferation medicine.
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| CN103566023A (en) * | 2013-10-29 | 2014-02-12 | 山东省立医院 | Preparation method and application of toothache anti-inflammation tablets |
| CN103705577A (en) * | 2013-12-30 | 2014-04-09 | 科创控股集团有限公司 | Antipyretic cold tablet crude drug volatile oil and extraction method thereof |
| CN105902650A (en) * | 2016-06-24 | 2016-08-31 | 中国人民解放军第三〇七医院 | Qingnaopian and preparation method thereof |
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| CN102397461A (en) * | 2011-11-27 | 2012-04-04 | 苏州派腾生物医药科技有限公司 | Preparation method of tablet for women's health and tranquilness |
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| CN102397461A (en) * | 2011-11-27 | 2012-04-04 | 苏州派腾生物医药科技有限公司 | Preparation method of tablet for women's health and tranquilness |
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| Title |
|---|
| 药典委员会: "《***部颁标准(中药成方制剂)》", 31 December 1991, article "清眩片", pages: Z4-183 * |
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| CN103566023A (en) * | 2013-10-29 | 2014-02-12 | 山东省立医院 | Preparation method and application of toothache anti-inflammation tablets |
| CN103566023B (en) * | 2013-10-29 | 2015-11-18 | 山东省立医院 | A kind of preparation method of toothache Pianomide and application |
| CN103705577A (en) * | 2013-12-30 | 2014-04-09 | 科创控股集团有限公司 | Antipyretic cold tablet crude drug volatile oil and extraction method thereof |
| CN105902650A (en) * | 2016-06-24 | 2016-08-31 | 中国人民解放军第三〇七医院 | Qingnaopian and preparation method thereof |
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