CN102988723B - Preparation method and application of traditional Chinese medicine - Google Patents
Preparation method and application of traditional Chinese medicine Download PDFInfo
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- CN102988723B CN102988723B CN201210378459.6A CN201210378459A CN102988723B CN 102988723 B CN102988723 B CN 102988723B CN 201210378459 A CN201210378459 A CN 201210378459A CN 102988723 B CN102988723 B CN 102988723B
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Abstract
The invention provides a preparation method of a yin tonifying and calming tablet, which is prepared from crude drugs including 75g of angelica sinensis, 57g of radix ophiopogonis, 48g of schisandra chinensis, 39g of vine of multiflower knotweed, 39g of rhizome rehmanniae, 75g of poria cocos, 18g of platycladi seed, 75g of codonopsis pilosula, 93g of concha margaritiferausta, 57g of radix scrophulariae, 57g of salvia miltiorrhiza, 39g of polygala tenuifolia, 39g of platycodon grandiflorum and 10.2g of cinnabar through supercritical extraction and microwave extraction, so that the content of ferulic acid is greatly improved. The invention also provides the application of the yin tonifying and calming tablet in preparing a medicine for restraining proliferation of mice leukemia cell, L1210 cell.
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 Yang Yin Zhen Jing Tablet.
Background technology
Yang Yin Zhen Jing Tablet is recorded in Ministry of Public Health standard WS3-B-0360-90, by Radix Angelicae Sinensis 75g, Radix Ophiopogonis, 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g make as crude drug, energy nourishing YIN and benefiting blood, tranquillizing the mind by relieving convulsion.For deficiency of heart-blood, god seized with terror is forgetful, vexed uneasiness, palpitation and insomnia.
In prior art, not yet there is Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet.
Another object of the present invention is to provide a kind of Yang Yin Zhen Jing Tablet to suppress the application in mouse leukemia cell L1210 cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of Yang Yin Zhen Jing Tablet, by Radix Angelicae Sinensis 75g, Radix Ophiopogonis, 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g make as crude drug, described method is comprised of the following step: get Radix Angelicae Sinensis, Semen Platycladi, join in 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, 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 1000, every heavy 0.3g.
The preparation method of above-mentioned a kind of Yang Yin Zhen Jing Tablet, 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 Yang Yin Zhen Jing Tablet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of Yang Yin Zhen Jing Tablet, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned Yang Yin Zhen Jing Tablet suppresses the application in mouse leukemia cell L1210 cell proliferation medicine in preparation.
In prior art, every 0.5g of Yang Yin Zhen Jing Tablet, each 4-6 sheet, 3 times on the one, the every 0.3g of Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet prepared by test one, distinct methods
1, instrument and reagent Yang Yin Zhen Jing Tablet of the present invention: press embodiment 3 method preparations, use 720g crude drug, make 1000 through extracting, every heavy 0.3g.Former Yang Yin Zhen Jing Tablet, by the preparation of ministry standard method, is used 720g crude drug, through extracting, makes 1000, every heavy 0.3g.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 Yang Yin Zhen Jing Tablet of the present invention and former Yang Yin Zhen Jing Tablet, porphyrize, mixes, and gets 1g, accurately weighed, 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 Yang Yin Zhen Jing Tablet of the present invention, the content of ferulic acid is 0.98mg/ sheet; And the content of ferulic acid is 0.19mg/ sheet in former Yang Yin Zhen Jing Tablet, in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned research shows, the Yang Yin Zhen Jing Tablet that adopts the present invention to prepare, and active constituent content is higher than the standby Yang Yin Zhen Jing Tablet 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 Angelicae Sinensis 75g, Radix Ophiopogonis 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g, by Radix Angelicae Sinensis, Semen Platycladi, join in 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, 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 1000, every heavy 0.3g.
After testing, in finished product, the content of ferulic acid is 1.11mg/ sheet.
Embodiment 2
Get Radix Angelicae Sinensis 75g, Radix Ophiopogonis 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g, by Radix Angelicae Sinensis, Semen Platycladi, 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 3m1/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 1000, every heavy 0.3g.
After testing, in finished product, the content of ferulic acid is 1.05mg/ sheet.
Embodiment 3
Get Radix Angelicae Sinensis 75g, Radix Ophiopogonis 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g, by Radix Angelicae Sinensis, Semen Platycladi, 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 2m1/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 1000, every heavy 0.3g.
After testing, in finished product, the content of ferulic acid is 0.98mg/ sheet.
Embodiment 4: Yang Yin Zhen Jing Tablet suppresses the experimentation data of L1210 cell proliferation
1 experiment material
1.1 experiment cell strains
Mouse leukemia cell L1210 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Yang Yin Zhen Jing Tablet of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg Yang Yin Zhen Jing Tablet, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe packing ,-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); 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/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) L1210 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, 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%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add Yang Yin Zhen Jing Tablet 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 L1210 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 Yang Yin Zhen Jing Tablet affects and grinds L1210 cell inhibitory effect
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Yang Yin Zhen Jing Tablet can suppress L1210 cell proliferation, reduces the Growth of Cells number of L1210 cell, and this effect is dose dependent.
Claims (4)
1. a Chinese patent medicine suppresses the application in mouse leukemia cell L1210 cell proliferation medicine in preparation, it is characterized in that Chinese patent medicine by Radix Angelicae Sinensis 75g, Radix Ophiopogonis 57g, Fructus Schisandrae Chinensis 48g, Caulis Polygoni Multiflori 39g, Radix Rehmanniae 39g, Poria 75g, Semen Platycladi 18g, Radix Codonopsis 75g, Concha Margaritifera 93g, Radix Scrophulariae 57g, Radix Salviae Miltiorrhizae 57g, Radix Polygalae 39g, Radix Platycodonis 39g, Cinnabaris 10.2g as crude drug, make, preparation method is comprised of the following step: get Radix Angelicae Sinensis, Semen Platycladi, 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 1-3m1/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 1000, every heavy 0.3g.
2. a kind of Chinese patent medicine suppresses the application in mouse leukemia cell L1210 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in Chinese patent medicine preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. a kind of Chinese patent medicine suppresses the application in mouse leukemia cell L1210 cell proliferation medicine in preparation according to claim 1, it is characterized in that the 500W of microwave extracting power described in Chinese patent medicine preparation method, extracts 6 minutes at every turn.
4. a kind of Chinese patent medicine suppresses the application in mouse leukemia cell L1210 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in Chinese patent medicine 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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| CN1634352A (en) * | 2004-10-11 | 2005-07-06 | 任连智 | Compound diabetes-treating sugar-decreasing fat-reducing preparation and preparation method thereof |
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