CN102988723A - Preparation method and application of yin tonifying and calming tablet - Google Patents

Preparation method and application of yin tonifying and calming tablet Download PDF

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CN102988723A
CN102988723A CN2012103784596A CN201210378459A CN102988723A CN 102988723 A CN102988723 A CN 102988723A CN 2012103784596 A CN2012103784596 A CN 2012103784596A CN 201210378459 A CN201210378459 A CN 201210378459A CN 102988723 A CN102988723 A CN 102988723A
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radix
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Huang Chuanfeng
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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

A kind of preparation method of Yang Yin Zhen Jing Tablet and application
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 Yang Yin Zhen Jing Tablet.
Background technology
Yang Yin Zhen Jing Tablet is recorded in Ministry of Public Health standard WS3-B-0360-90,57g, 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 by Radix Angelicae Sinensis 75g, Radix Ophiopogonis, the energy nourishing YIN and benefiting blood, tranquillizing the mind by relieving convulsion.Be used for deficiency of heart-blood, god seized with terror is forgetful, vexed uneasiness, palpitation and insomnia.
In the prior art, not yet there is Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet.
Another object of the present invention is to provide the application of a kind of Yang Yin Zhen Jing Tablet in preparation inhibition mouse leukemia cell L1210 cell proliferation medicine.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of preparation method of Yang Yin Zhen Jing Tablet, 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 by Radix Angelicae Sinensis 75g, Radix Ophiopogonis, described method is comprised of the following step: get Radix Angelicae Sinensis, Semen Platycladi, 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 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 the 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.
The application of above-mentioned Yang Yin Zhen Jing Tablet in preparation inhibition mouse leukemia cell L1210 cell proliferation medicine.
In the 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, 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 Yang Yin Zhen Jing Tablet of test one, distinct methods preparation
1, instrument and reagent Yang Yin Zhen Jing Tablet of the present invention: press the preparation of embodiment 3 methods, use the 720g crude drug, make 1000 through extraction, every heavy 0.3g.Former Yang Yin Zhen Jing Tablet by the preparation of ministry standard method, uses the 720g crude drug, makes 1000 through extraction, every heavy 0.3g.Agilent 1200 high performance liquid chromatographs; 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 Yang Yin Zhen Jing Tablet of the present invention and former Yang Yin Zhen Jing Tablet, 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 0.98mg/ sheet in the Yang Yin Zhen Jing Tablet of the present invention; And the content of ferulic acid is the 0.19mg/ sheet in the former Yang Yin Zhen Jing Tablet, and in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned studies show that, the Yang Yin Zhen Jing Tablet that adopts the present invention to prepare, 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 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 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, with Radix Angelicae Sinensis, Semen Platycladi, 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 1000, every heavy 0.3g.
After testing, the content of ferulic acid is the 1.11mg/ sheet in the finished product.
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, with Radix Angelicae Sinensis, Semen Platycladi, 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 1000, every heavy 0.3g.
After testing, the content of ferulic acid is the 1.05mg/ sheet in the finished product.
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, with Radix Angelicae Sinensis, Semen Platycladi, 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 1000, every heavy 0.3g.
After testing, the content of ferulic acid is the 0.98mg/ sheet in the finished product.
Embodiment 4: Yang Yin Zhen Jing Tablet suppresses the experimentation data of L1210 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing the 100mg Yang Yin Zhen Jing Tablet, 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.12100-061 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.11810-033 Lot.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: SPECTRAMAX 190); 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/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 (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 L1210 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 3ml 0.25% 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 Yang Yin Zhen Jing Tablet 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 Microsoft Excel 2003 softwares, 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 L1210 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 Yang Yin Zhen Jing Tablet affects the L1210 cell inhibitory effect and grinds
Figure BDA00002230035800051
Figure BDA00002230035800052
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
Yang Yin Zhen Jing Tablet can suppress the L1210 cell proliferation, reduces the Growth of Cells number of L1210 cell, and this effect is dose dependent.

Claims (5)

1. the preparation method of a Yang Yin Zhen Jing Tablet, 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 by Radix Angelicae Sinensis 75g, Radix Ophiopogonis, it is characterized in that described method is comprised of the following step: get Radix Angelicae Sinensis, Semen Platycladi, 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 1000, every heavy 0.3g.
2. the preparation method of described a kind of Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet 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 Yang Yin Zhen Jing Tablet in preparation inhibition mouse leukemia cell L1210 cell proliferation medicine.
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CN104686750A (en) * 2015-01-30 2015-06-10 苏州市天灵中药饮片有限公司 Traditional Chinese medicine health tea for tranquilization and preparation method thereof
CN105878886A (en) * 2014-12-17 2016-08-24 杨少梅 Medicine for treating sleep disorder

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