CN102988667B - Preparation method and application of tablet for regulating menstruation and activating blood - Google Patents
Preparation method and application of tablet for regulating menstruation and activating blood Download PDFInfo
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- CN102988667B CN102988667B CN201210388267.3A CN201210388267A CN102988667B CN 102988667 B CN102988667 B CN 102988667B CN 201210388267 A CN201210388267 A CN 201210388267A CN 102988667 B CN102988667 B CN 102988667B
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a preparation method of a tablet for regulating menstruation and activating blood. The tablet is prepared by using 20g of costustoot, 20g of szechuan lovage rhizome, 20g of corydalis yanhusuo processed with rice vinegar, 60g of Chinese angelica, 40g of cooked rehmannia, 40g of red paeony root, 30g of safflower, 30g of combined spicebush root, 30g of Largehead atractylodes rhizome, 60g of danshen root, 60g of processed cyperus, 10g of medcinal evodia fruit processed with licorice water, 60g of hiraute shiny bugleweed herb, 60g of suberect spatholobus stems and 80g of Chinese dodder seeds as bulk drugs and adopting supercritical extraction and microwave-assisted extraction modes. By adopting the preparation method, the ferulic acid content is greatly improved; and the invention also provides application of the tablet for regulating menstruation and activating blood in preparation of drugs for inhibiting cell proliferation of mice mast cell carcinoma P815.
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 regulating menstruation and activating blood sheet.
Background technology
Regulating menstruation and activating blood sheet is recorded in Ministry of Public Health standard WS3-B-0614-91, by Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, as crude drug, made, energy regulating menstruation and activating blood, promoting the circulation of QI to relieve pain.For menoxenia, menalgia.
In prior art, not yet there is regulating menstruation and activating blood sheet 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 regulating menstruation and activating blood sheet.
Another object of the present invention is to provide the application of a kind of regulating menstruation and activating blood sheet in the medicine of preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of regulating menstruation and activating blood sheet, by Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, as crude drug, made, described method is comprised of the following step: get the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 1-3ml/(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 600, every heavy 0.5g.
The preparation method of above-mentioned a kind of regulating menstruation and activating blood sheet, 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 regulating menstruation and activating blood sheet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of regulating menstruation and activating blood sheet, 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 regulating menstruation and activating blood sheet in the medicine of preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation.
In prior art, every 0.5g of regulating menstruation and activating blood sheet, each 5,3 times on the one, the every 0.5g of regulating menstruation and activating blood sheet that adopts the present invention to be prepared into only needs 3 at every turn, within 1st, takes 3 times, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of ferulaic acid content in regulating menstruation and activating blood sheet prepared by test one, distinct methods
1, instrument and reagent regulating menstruation and activating blood sheet of the present invention: press embodiment 3 method preparations, use 620g crude drug, make 600 through extracting, every heavy 0.5g.Former regulating menstruation and activating blood sheet, by the preparation of ministry standard method, is used 620g crude drug, through extracting, makes 600, every heavy 0.5g.Agilent1200 high performance liquid chromatograph; 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; Detection wavelength is 280nm.Number of theoretical plate is pressed 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 every 1ml containing the solution of 18 μ g, obtains.
The preparation of need testing solution: get regulating menstruation and activating blood sheet of the present invention and former regulating menstruation and activating blood sheet, porphyrize, mixes, and gets 1g, accurately weighed, and 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 regulating menstruation and activating blood sheet of the present invention, the content of ferulic acid is 1.38mg/ sheet; And the content of ferulic acid is 0.29mg/ sheet in former regulating menstruation and activating blood sheet, in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned research shows, the regulating menstruation and activating blood sheet that adopts the present invention to prepare, and active constituent content is higher than the standby regulating menstruation and activating blood sheet 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 Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, by the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, join CO
2in 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 1ml/(g crude drug min), extraction time 150min, 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 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 600, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is 1.41mg/ sheet.
Embodiment 2
Get Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, by the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 3ml/(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 600, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is 1.45mg/ sheet.
Embodiment 3
Get Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, by the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 2ml/(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 600, every heavy 0.5g.
After testing, in finished product, the content of ferulic acid is 1.38mg/ sheet.
Embodiment 4: regulating menstruation and activating blood sheet suppresses the experimentation data of P815 cell proliferation
1 experiment material
1.1 experiment cell strains
Mouse hypertrophy cell cancer P815 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: regulating menstruation and activating blood sheet of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg regulating menstruation and activating blood sheet, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ ldoff 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.12100-061Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The NaHCO3(Shanghai Jiu Yi chemical reagent Cat.No.11810-033Lot.No.1088387 of 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: SPECTRA MAX190); CO
2incubator (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/N020579); 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) P815 cell uses DMEM+10%FBS in 37 ℃, 5%CO
2carry out cellar culture (10cm culture dish), when Growth of Cells is during to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml0.25% 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%CO
2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add regulating menstruation and activating blood sheet 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 Excel2003 software, 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 P815 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 regulating menstruation and activating blood sheet is on P815 cell inhibitory effect impact research
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Regulating menstruation and activating blood sheet can suppress P815 cell proliferation, reduces the Growth of Cells number of P815 cell, and this effect is dose dependent.
Claims (4)
1. a regulating menstruation and activating blood sheet suppresses the application in the medicine of mouse hypertrophy cell cancer P815 cell proliferation in preparation, it is characterized in that regulating menstruation and activating blood sheet made as crude drug by Radix Aucklandiae 20g, Rhizoma Chuanxiong 20g, Rhizoma Corydalis (processed with vinegar) 20g, Radix Angelicae Sinensis 60g, Radix Rehmanniae Preparata 40g, Radix Paeoniae Rubra 40g, Flos Carthami 30g, Radix Linderae 30g, Rhizoma Atractylodis Macrocephalae 30g, Radix Salviae Miltiorrhizae 60g, Rhizoma Cyperi (processed) 60g, liquorice beverage Fructus Evodiae (processed) 10g, Herba Lycopi 60g, Caulis Spatholobi 60g, Semen Cuscutae 80g, preparation method is comprised of the following step: get the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 1-3ml/(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 600, every heavy 0.5g.
2. the application of a kind of regulating menstruation and activating blood sheet in the medicine of preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation according to claim 1, is characterized in that CO described in preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. the application of a kind of regulating menstruation and activating blood sheet in the medicine of preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation according to claim 1, is characterized in that the power of microwave extracting described in preparation method 500W, extracts 6 minutes at every turn.
4. the application of a kind of regulating menstruation and activating blood sheet in the medicine of preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation according to claim 1, 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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| CN103301399A (en) * | 2013-05-11 | 2013-09-18 | 闫玉枝 | Traditional Chinese medicine for treating pelvic inflammation |
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| CN103705683A (en) * | 2013-12-22 | 2014-04-09 | 青岛琴诚医药技术有限公司 | Preparation method and application of Sixiao pills |
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| CN104055998A (en) * | 2014-07-06 | 2014-09-24 | 杨天梅 | Menstruation regulating and kidney reinforcing soup |
| CN104922227A (en) * | 2015-05-28 | 2015-09-23 | 吉林省正和药业集团股份有限公司 | Preparation method of Yuanhe stomach tablet and application |
| CN104922375A (en) * | 2015-06-01 | 2015-09-23 | 太仓思瑞生物科技有限公司 | Traditional Chinese medicine patch for treating irregular menstruation |
| CN104906307A (en) * | 2015-06-01 | 2015-09-16 | 太仓思瑞生物科技有限公司 | Application of traditional Chinese medicine externally applied for treating irregular menstruation |
| CN107536885A (en) * | 2017-10-07 | 2018-01-05 | 南京正宽医药科技有限公司 | A kind of hemostatic traditional Chinese medicine composition and preparation method thereof |
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