CN102836381A - Preparation method and application of middle calming tablet - Google Patents
Preparation method and application of middle calming tablet Download PDFInfo
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- CN102836381A CN102836381A CN201210375789XA CN201210375789A CN102836381A CN 102836381 A CN102836381 A CN 102836381A CN 201210375789X A CN201210375789X A CN 201210375789XA CN 201210375789 A CN201210375789 A CN 201210375789A CN 102836381 A CN102836381 A CN 102836381A
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Abstract
The invention provides a preparation method of a middle calming tablet. The middle calming tablet is prepared from 180g of cassia twig, 180g of vinegar-treated corydalis tuber, 180g of calcined oyster, 120g of common fennel, 120g of amomum fruit, 60g of Chinese ginger and 120g of liquoric root serving as bulk pharmaceuticals. The middle calming tablet is prepared through ultrafine extraction and microwave extraction, so that the convent of tetrahydropalmatine is increased greatly. The invention further provides an application of the middle calming tablet to preparation of a medicament for inhibiting cell proliferation of rat breast cancer cells SHZ-88.
Description
Technical field
The present invention relates to the Chinese medicine preparation technical field, be specifically related to a kind of method for preparing and application of regulating the spleen and stomach sheet.
Background technology
The regulating the spleen and stomach sheet is recorded in Ministry of Public Health standard WS3-B-0929-91; Process as crude drug by Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g; The ability warming spleen and stomach for dispelling cold, regulating QI to relieve pain, stomach function regulating preventing or arresting vomiting.Be used for gastralgia, chronic gastritis, hyperchlorhydria, stomach and duodenal ulcer.
In the prior art, do not have the regulating the spleen and stomach sheet extracting the report that adopts supercritical and microwave technology aspect the preparation as yet, and the method that powder and decocting boil is beaten in employing; Technology is coarse, backward, and impurity is many, causes patient's consumption excessive; Be inconvenient to take, had a strong impact on these article and used clinically.
Summary of the invention
Goal of the invention:, the object of the present invention is to provide a kind of method for preparing of regulating the spleen and stomach sheet in order to address the above problem.
Another object of the present invention is to provide a kind of regulating the spleen and stomach sheet to suppress the application in the rat breast cancer cell SHZ-88 cell proliferation medicine in preparation.
Technical scheme: the objective of the invention is to realize through following scheme:
A kind of method for preparing of regulating the spleen and stomach sheet is processed as crude drug by Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g, and described method is made up of the following step: get Ramulus Cinnamomi, Rhizoma Corydalis (processed with vinegar), Fructus Foeniculi, Fructus Amomi, Rhizoma Alpiniae Officinarum; Join in the 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 gets supercritical extract, and is 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, and each 4-8 minute, combining extraction liquid; Concentrate, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents; Decompression recycling ethanol concentrates and drying, gets the microwave extraction thing, and is subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol system granule, drying, tabletting is processed 1000, every heavy 0.5g.
The method for preparing of above-mentioned a kind of regulating the spleen and stomach sheet, said CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
The method for preparing of above-mentioned a kind of regulating the spleen and stomach sheet, said microwave extracting power 500W extracted 6 minutes at every turn.
The method for preparing of above-mentioned a kind of regulating the spleen and stomach sheet, said CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned regulating the spleen and stomach sheet suppresses the application in the rat breast cancer cell SHZ-88 cell proliferation medicine in preparation.
In the prior art, every 0.5g of regulating the spleen and stomach sheet, each 4-6 sheet, 3 times on the one, the every 0.5g of regulating the spleen and stomach sheet that adopts the present invention to be prepared into only needs 3 at every turn, takes 3 times in 1st, has significantly reduced dose having under the condition of more active component.This conclusion can be through following evidence.
The comparison of tetrahydropalmatine content in the regulating the spleen and stomach sheet of test one, distinct methods preparation
1, instrument and reagent regulating the spleen and stomach sheet of the present invention: press the preparation of embodiment 3 methods, use the 1000g crude drug, process 1000, every heavy 0.5g through extracting.Former regulating the spleen and stomach sheet by the preparation of ministry standard method, uses the 100g crude drug, processes 1000 through extracting, every heavy 0.5g.The Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Tetrahydropalmatine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Chromatographic condition and system suitability test: use octadecylsilane chemically bonded silica to be filler; Methanol-water-phosphoric acid (40:60:0.2) is a mobile phase; The detection wavelength is 280nm.Number of theoretical plate is pressed the tetrahydropalmatine peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours tetrahydropalmatine reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methanol and processes the solution that every 1ml contains 18 μ g, promptly gets.
The preparation of need testing solution: get regulating the spleen and stomach sheet of the present invention and former regulating the spleen and stomach sheet, porphyrize, mixing is got 1g, and accurate the title, decide, the accurate 70% ethanol 20ml that adds, close plug, supersound process 10 minutes, centrifugal, get supernatant, promptly get.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and promptly get.
3, result
The result shows that the content of tetrahydropalmatine is the 1.88mg/ sheet in the regulating the spleen and stomach sheet of the present invention; And the content of tetrahydropalmatine is the 0.59mg/ sheet in the former regulating the spleen and stomach sheet, and under the situation that dose reduces, tetrahydropalmatine content improves a lot.
Above-mentioned research shows, adopts the regulating the spleen and stomach sheet of the present invention's preparation, and active constituent content is higher than the regulating the spleen and stomach sheet of ministry standard method preparation.
The specific embodiment
Below through the embodiment form; Foregoing of the present invention is remake further detailed description; But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g; With Ramulus Cinnamomi, Rhizoma Corydalis (processed with vinegar), Fructus Foeniculi, Fructus Amomi, Rhizoma Alpiniae Officinarum, 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 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, and each 4 minutes, combining extraction liquid; Concentrate, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents; Decompression recycling ethanol concentrates and drying, gets the microwave extraction thing, and is subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol system granule, drying, tabletting is processed 1000, every heavy 0.5g.
Through detecting, the content of tetrahydropalmatine is the 1.94mg/ sheet in the finished product.
Embodiment 2
Get Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g,, join CO Ramulus Cinnamomi, Rhizoma Corydalis (processed with vinegar), Fructus Foeniculi, Fructus Amomi, Rhizoma Alpiniae Officinarum
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, 50 ℃ of temperature, CO
2Flow 3m1/g crude drug min, extraction time 180min gets supercritical extract, and is 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, and each 8 minutes, combining extraction liquid; Concentrate, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents; Decompression recycling ethanol concentrates and drying, gets the microwave extraction thing, and is subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol system granule, drying, tabletting is processed 1000, every heavy 0.5g.
Through detecting, the content of tetrahydropalmatine is the 1.91mg/ sheet in the finished product.
Embodiment 3
Get Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g,, join CO Ramulus Cinnamomi, Rhizoma Corydalis (processed with vinegar), Fructus Foeniculi, Fructus Amomi, Rhizoma Alpiniae Officinarum
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 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, and each 6 minutes, combining extraction liquid; Concentrate, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents; Decompression recycling ethanol concentrates and drying, gets the microwave extraction thing, and is subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol system granule, drying, tabletting is processed 1000, every heavy 0.5g.
Through detecting, the content of tetrahydropalmatine is the 1.88mg/ sheet in the finished product.Embodiment 4: the regulating the spleen and stomach sheet suppresses the experimentation data of SHZ-88 cell proliferation
1 experiment material
1.1 cell strain is used in experiment
Rat breast cancer cell SHZ-88 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS is conventional to be cultivated.
1.2 experiment medicine
Research medicine: regulating the spleen and stomach sheet of the present invention: press the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing 100mg regulating the spleen and stomach sheet, 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 the usefulness of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
DMEM (Cat.No.12100-061Lot.No.758137 of 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); PBS (laboratory autogamy); 1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); It is thus clear that-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX190); CO2 incubator (FORMA model: 3111); Super-clean bench (the safe and sound manufactured model of Su Jing group: SW-CJ-ZFD); Pure water appearance (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); Desk-top electric drying oven with forced convection (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 SHZ-88 cell carries out routine with DMEM+10%FBS in 37 ℃, 5%CO2 and cultivates (10cm culture dish), when cell grows to logarithmic (log) phase, and collecting cell; Discard culture fluid, PBS fine laundering 3 times adds 3ml0.25% trypsin-0.04%EDTA; Behind 37 ℃ of digestion 2min,, behind the piping and druming cell it is changed in the centrifuge tube to wherein adding 5ml complete medium neutralization reaction; The centrifugal 5min of 1000rpm, 3 * 104/ml of adjustment concentration of cell suspension.2) the cell kind is gone in 96 well culture plates, every hole adds cell suspension 180 μ l, and culture plate is put into cell culture incubator, and (37 ℃, 5%CO2) routine is cultivated.
3) according to the cell growing state, generally grow to 50%-70%, add regulating the spleen and stomach 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, claps driedly 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), 6 multiple holes of every settings.
7) result representes with the suppression ratio of medicine pair cell: cell increment suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value * 100%.Experiment repetition 3 times.
3 statistical dispositions
Adopt correlation analysis and Student t check in the Microsoft Excel2003 software, data are represented with mean ± S.D..
4 experimental results
Mtt assay experiment back statistical result showed; Compare with matched group; When dosage reaches 5mg/ml; To SHZ-88 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 the spleen and stomach sheet is to SHZ-88 cell inhibitory effect influence research
Annotate: compare * P < 0.01 with matched group; * P < 0.001
5 experiment conclusion
The regulating the spleen and stomach sheet can suppress the SHZ-88 cell proliferation, reduces the cell growing number of SHZ-88 cell, and this effect is dose dependent.
Claims (5)
1. the method for preparing of a regulating the spleen and stomach sheet; Process as crude drug by Ramulus Cinnamomi 180g, Rhizoma Corydalis (processed with vinegar) 180g, Concha Ostreae (calcined) 180g, Fructus Foeniculi 120g, Fructus Amomi 120g, Rhizoma Alpiniae Officinarum 60g, Radix Glycyrrhizae 120g; It is characterized in that described method is made up of the following step: get Ramulus Cinnamomi, Rhizoma Corydalis (processed with vinegar), Fructus Foeniculi, Fructus Amomi, Rhizoma Alpiniae Officinarum, 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 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, and each 4-8 minute, combining extraction liquid; Concentrate, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents; Decompression recycling ethanol concentrates and drying, gets the microwave extraction thing, and is subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol system granule, drying, tabletting is processed 1000, every heavy 0.5g.
2. according to the method for preparing of the said a kind of regulating the spleen and stomach sheet of claim 1, it is characterized in that said CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
3. according to the method for preparing of the said a kind of regulating the spleen and stomach sheet of claim 1, it is characterized in that said microwave extracting power 500W, extracted 6 minutes at every turn.
4. according to the method for preparing of the said a kind of regulating the spleen and stomach sheet of claim 1, it is characterized in that said CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
5. suppress the application in the rat breast cancer cell SHZ-88 cell proliferation medicine according to the said a kind of regulating the spleen and stomach sheet of claim 1 in preparation.
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Cited By (3)
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| CN103536691A (en) * | 2013-10-31 | 2014-01-29 | 张芝莲 | Preparation method and application of pills containing schisandra chinensis |
| CN103566023A (en) * | 2013-10-29 | 2014-02-12 | 山东省立医院 | Preparation method and application of toothache anti-inflammation tablets |
| CN104288660A (en) * | 2014-10-16 | 2015-01-21 | 济南新起点医药科技有限公司 | Preparation method of fomes officinalis cough and asthma treating tablets and application of fomes officinalis cough and asthma treating tablets in preparation of medicine for inhibiting cell proliferation of mouse lymphoma cell EL4 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN103536691A (en) * | 2013-10-31 | 2014-01-29 | 张芝莲 | Preparation method and application of pills containing schisandra chinensis |
| CN104288660A (en) * | 2014-10-16 | 2015-01-21 | 济南新起点医药科技有限公司 | Preparation method of fomes officinalis cough and asthma treating tablets and application of fomes officinalis cough and asthma treating tablets in preparation of medicine for inhibiting cell proliferation of mouse lymphoma cell EL4 |
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