CN102988502B - Preparation method and application of Desheng tablet - Google Patents
Preparation method and application of Desheng tablet Download PDFInfo
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- CN102988502B CN102988502B CN201210390320.3A CN201210390320A CN102988502B CN 102988502 B CN102988502 B CN 102988502B CN 201210390320 A CN201210390320 A CN 201210390320A CN 102988502 B CN102988502 B CN 102988502B
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Abstract
The invention provides a preparation method and applications of a Desheng tablet. The Desheng tablet is prepared from bulk drugs, including 960g of leonurus japonicus, 160g of radix bupleuri, 320g of angelica sinensis, 80g of ligusticum wallichii, 320g of white paeony root and 80g of costustoot, by supercritical extraction and microwave extraction. The invention also provides an application of the Desheng tablet in preparation of drugs for inhibiting proliferation of A-204 cells of rhabdomyosarcoma.
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 that obtains raw cook.
Background technology
Obtain raw cook and be recorded in Ministry of Public Health standard WS3-B-0623-91, made as crude drug by Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, energy regulating menstruation and nourishing blood, activating QI to eliminate phlegm.For menoxenia, abdominal pain in menstruation, stagnation of QI and blood, mass in the abdomen.
In prior art, not yet have to such an extent that raw cook adopts 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 that obtains raw cook.
It is a kind of that raw cook suppresses the application in rhabdomyosarcoma A-204 cell proliferation medicine in preparation that another object of the present invention is to provide.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of preparation method that obtains raw cook, by Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, as crude drug, made, described method is comprised of the following step: get Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, join in CO2 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, 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 400-600W, extract 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
Above-mentioned a kind of preparation method that obtains raw cook, described CO
2the percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
Above-mentioned a kind of preparation method that obtains raw cook, described microwave extracting power 500W extracts 6 minutes at every turn.
Above-mentioned a kind of preparation method that obtains raw cook, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned that raw cook suppresses the application in A-204 cell proliferation medicine in preparation.
In prior art, obtain every 0.5g of raw cook, each 4,2 times on the one, adopt the present invention to be prepared into every 0.5g of raw cook, only need 2 at every turn, within 1st, take 2 times, greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison that obtains ferulaic acid content in raw cook prepared by test one, distinct methods
1, instrument and reagent the present invention obtain raw cook: press embodiment 3 method preparations, use the 1920g crude drug, through extraction, make 1000, every heavy 0.5g.The former raw cook that obtains, by the preparation of ministry standard method, used the 1920g crude drug, through extraction, makes 1700, 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: with octadecylsilane chemically bonded silica, be filler; 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 at 60 ℃ of drying under reduced pressure ferulic acid reference substance of 4 hours appropriate, adds methanol and makes the solution of every 1ml containing 18 μ g, obtains.
The preparation of need testing solution: get the present invention and obtain raw cook and the former raw cook that obtains, porphyrize, mix, and gets 1g, accurately weighed, and precision adds 70% ethanol 20ml, close plug, and 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 the injection liquid chromatography, measure, and obtains.
3, result
Result shows, the content that the present invention obtains ferulic acid in raw cook is the 1.46mg/ sheet; And the former content that obtains ferulic acid in raw cook is the 0.35mg/ sheet, in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned research shows, adopt the present invention to prepare raw cook, active constituent content standby higher than the ministry standard legal system raw cook.
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 realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, by Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, 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, CO
2flow 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, extract 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is the 1.55mg/ sheet.
Embodiment 2
Get Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, by Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, 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, 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 600W, extract 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is the 1.54mg/ sheet.
Embodiment 3
Get Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, by Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, 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, 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 500W, extract 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is the 1.46mg/ sheet.
Embodiment 4: obtain the experimentation data that raw cook suppresses the A-204 cell proliferation
1 experiment material
1.1 experiment cell strain
Rhabdomyosarcoma (A-204), Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention obtains raw cook: press embodiment 3 method preparations.
The medicinal liquid liquid storage: take 100mg and obtain raw cook, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe packing, and-20 ℃ of storages, 0.2 μ m filter filters the use of dehydrated alcohol in order to 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); 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) the A-204 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, discard 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 the piping and druming cell, it is proceeded in centrifuge tube, the centrifugal 5min of 1000rpm, adjust 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 to obtain raw cook solution, continue to cultivate 24h.
4) add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, the 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.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), set 6 multiple holes for every group.
7) with medicine, the suppression ratio to cell means result:
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 mean with mean ± S.D..
4 experimental results
Statistical result showed after the mtt assay experiment, with matched group, compare, when dosage reaches 5mg/ml, to A-204 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 obtains raw cook to A-204 cell inhibitory effect impact research
Annotate: with matched group, compare,
*p<0.01;
*p<0.001
5 experiment conclusion
Obtain raw cook and can suppress the A-204 cell proliferation, reduce the Growth of Cells number of A-204 cell, this effect is dose dependent.
Claims (4)
1. one kind obtains the application of raw cook in preparation inhibition rhabdomyosarcoma A-204 cell proliferation medicine, it is characterized in that to such an extent that raw cook is made as crude drug by Herba Leonuri 960g, Radix Bupleuri 160g, Radix Angelicae Sinensis 320g, Rhizoma Chuanxiong 80g, Radix Paeoniae Alba 320g, Radix Aucklandiae 80g, preparation method is comprised of the following step: get Radix Bupleuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, 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, 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 400-600W, extract 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
According to claim 1 raw cook suppresses the application in rhabdomyosarcoma A-204 cell proliferation medicine in preparation, it is characterized in that obtaining CO described in the preparation method of raw cook
2the percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
According to claim 1 raw cook suppresses the application in rhabdomyosarcoma A-204 cell proliferation medicine in preparation, it is characterized in that obtaining microwave extracting power 500W described in the preparation method of raw cook, extract 6 minutes at every turn.
According to claim 1 raw cook suppresses the application in rhabdomyosarcoma A-204 cell proliferation medicine in preparation, it is characterized in that obtaining CO described in the preparation method of raw cook
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210390320.3A CN102988502B (en) | 2012-10-15 | 2012-10-15 | Preparation method and application of Desheng tablet |
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| CN102988502B true CN102988502B (en) | 2014-01-08 |
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| CN103610794B (en) * | 2013-11-26 | 2015-07-15 | 青岛大学附属医院 | Preparation method and application of nerve-calming tablets |
| CN103784574B (en) * | 2014-01-28 | 2016-04-06 | 济南伟传信息技术有限公司 | A kind of Chinese medicine for oral administration preventing and treating hemolytic disease of newborn |
| CN105582132A (en) * | 2016-03-07 | 2016-05-18 | 南京多宝生物科技有限公司 | Preparation method and application of liquorice root formulation granules |
| CN105748542A (en) * | 2016-03-07 | 2016-07-13 | 南京多宝生物科技有限公司 | Preparation method and application of angelica sinensis formula granules |
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| CN102397451B (en) * | 2011-11-26 | 2013-06-19 | 苏州派腾生物医药科技有限公司 | Preparation method of cholagogic tablet |
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