CN102988667A - 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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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 the 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
The regulating menstruation and activating blood sheet is recorded in Ministry of Public Health standard WS3-B-0614-91, 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, the energy regulating menstruation and activating blood, promoting the circulation of QI to relieve pain.Be used for menoxenia, menalgia.
In the prior art, not yet there is the regulating menstruation and activating blood sheet 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 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 preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation medicine.
Technical scheme: the objective 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 makes as crude drug, 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 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 ℃, 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 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 the 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 preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation medicine.
In the 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, takes 3 times in 1st, 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 the regulating menstruation and activating blood sheet of test one, distinct methods preparation
1, instrument and reagent regulating menstruation and activating blood sheet of the present invention: press the preparation of embodiment 3 methods, use the 620g crude drug, make 600 through extraction, every heavy 0.5g.Former regulating menstruation and activating blood sheet by the preparation of ministry standard method, uses the 620g crude drug, makes 600 through extraction, every heavy 0.5g.The Agilent1200 high performance liquid chromatograph; The METTLERAE240 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 regulating menstruation and activating blood sheet of the present invention and former regulating menstruation and activating blood sheet, 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 ferulic acid is the 1.38mg/ sheet in the regulating menstruation and activating blood sheet of the present invention; And the content of ferulic acid is the 0.29mg/ sheet in the former regulating menstruation and activating blood sheet, and in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned studies show that, the regulating menstruation and activating blood sheet that adopts the present invention to prepare, 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 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 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, with the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, join in the CO2 supercritical extraction device, ethanol is as entrainer, 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 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 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 600, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.41mg/ sheet in the finished product.
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, with the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 600, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.45mg/ sheet in the finished product.
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, with the Radix Aucklandiae, Rhizoma Chuanxiong, Rhizoma Corydalis (processed with vinegar), Radix Angelicae Sinensis, Flos Carthami, 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 600, every heavy 0.5g.
After testing, the content of ferulic acid is the 1.38mg/ sheet in the finished product.
Embodiment 4: the regulating menstruation and activating blood sheet suppresses the experimentation data of P815 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing 100mg regulating menstruation and activating blood sheet, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ ldoff 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.12100-061Lot.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: SPECTRAMAX190); 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/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 (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 P815 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 3ml0.25% 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 regulating menstruation and activating blood sheet 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 the Microsoft Excel2003 software, 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 P815 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 regulating menstruation and activating blood sheet is on P815 cell inhibitory effect impact research
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
The regulating menstruation and activating blood sheet can suppress the P815 cell proliferation, reduces the Growth of Cells number of P815 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of a 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, it is characterized in that 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 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 600, every heavy 0.5g.
2. the preparation method of described a kind of regulating menstruation and activating blood sheet 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 regulating menstruation and activating blood sheet 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 regulating menstruation and activating blood sheet 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 regulating menstruation and activating blood sheet in preparation inhibition mouse hypertrophy cell cancer P815 cell proliferation medicine.
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| CN103301399A (en) * | 2013-05-11 | 2013-09-18 | 闫玉枝 | Traditional Chinese medicine for treating pelvic inflammation |
| CN103536725A (en) * | 2013-10-31 | 2014-01-29 | 刘美福 | Preparation method and application of priceless health restoring powder |
| CN103656600A (en) * | 2013-12-22 | 2014-03-26 | 青岛琴诚医药技术有限公司 | Preparation method and application of twelve body channel warming pellet |
| 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 |
| CN104055998A (en) * | 2014-07-06 | 2014-09-24 | 杨天梅 | Menstruation regulating and kidney reinforcing soup |
| 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 |
| 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 |
| 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 |
| 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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Cited By (11)
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|---|---|---|---|---|
| CN103301399A (en) * | 2013-05-11 | 2013-09-18 | 闫玉枝 | Traditional Chinese medicine for treating pelvic inflammation |
| CN103536725A (en) * | 2013-10-31 | 2014-01-29 | 刘美福 | Preparation method and application of priceless health restoring powder |
| CN103536725B (en) * | 2013-10-31 | 2015-09-09 | 义乌市绿美生物科技有限公司 | A kind of preparation method of priceless health restoring powder and application |
| CN103656600A (en) * | 2013-12-22 | 2014-03-26 | 青岛琴诚医药技术有限公司 | Preparation method and application of twelve body channel warming pellet |
| 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 |
| CN103656600B (en) * | 2013-12-22 | 2016-03-23 | 李梅 | A kind of preparation method of 12 WENJING WAN and application |
| 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 |
| 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 |
| 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 |
| 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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