CN103768514A - Preparation method of Danshitongli tablet and application of Danshitongli tablet in preparing drugs for inhibiting myeloma cell SP2/0 from cell proliferation - Google Patents
Preparation method of Danshitongli tablet and application of Danshitongli tablet in preparing drugs for inhibiting myeloma cell SP2/0 from cell proliferation Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and particularly provides a preparation method and application of a Danshitongli tablet. The preparation method comprises the following steps: by using 225g of Herba Lysimachiae, 225g of oriental wormwood, 90g of radix curcumae, 150g of dried orange peel, 45g of radix scutellariae, 35g of frankincense, 80g of mirabilite, 35 of white alum, 45g of rhubarb, 45g of rhizoma sparganii, 45g of gardenia, 25g of licorice and 35g of myrrh as active pharmaceutical ingredients, carrying out superfine grinding, supercritical extraction, microwave extraction and macroporous resin adsorption so that the hesperidin content is greatly enhanced. The invention also provides application of the Danshitongli tablet in preparing drugs for inhibiting myeloma cell SP2/0 from cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of cholelithiasis treating Danshitongli tablet and suppress the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
Background technology
DanShiTongLi capsules standard No. WS-10324(ZD-0324)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle.Made as crude drug by Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g, there is clearing heat secreting bile, blood stasis dispelling calculus removing effect.For the acute and chronic cholecystitis due to dampness-heat in the liver and gallbladder, cholelithiasis etc.
In prior art, not yet there is DanShiTongLi capsules adopting micronizing, CO aspect extraction preparation
2the report that supercritical extraction, microwave technology and macroporous adsorption resin technology extract, and Chinese medicine is directly beaten the method that powder is got, decocting boils, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, is inconvenient to take, and has had a strong impact on this product and has applied clinically.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of cholelithiasis treating Danshitongli tablet.
Another object of the present invention is to provide a kind of cholelithiasis treating Danshitongli tablet to suppress the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
The object of the invention is to realize by following scheme:
A kind of preparation method of cholelithiasis treating Danshitongli tablet, be made up as crude drug of Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g, described preparation method is made up of the following step: Natrii Sulfas, Alumen micronizing become the fine powder of particle size range 5-45 μ m for subsequent use, get Olibanum, Myrrha, be ground into fine powder, 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 180-220min, obtains supercritical extract, for subsequent use, Olibanum after extraction, Myrrha residue and other nine tastes medical materials, pulverize, add 70% ethanol of 3.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, microwave extraction thing mixes and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2in supercritical extraction, extracting pressure is 25MPa.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2in supercritical extraction, extraction temperature is 60 ℃.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described microwave extracting power is 700W.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned cholelithiasis treating Danshitongli tablet suppresses the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
In prior art, DanShiTongLi capsules is oral, one time 5,3 times on the one.DanShiTongLi capsules dosage is large.The every heavy 0.3g of cholelithiasis treating Danshitongli tablet that adopts the inventive method to be prepared into only needs 2 at every turn, within 1st, takes 3 times.Under the condition with more active component, greatly reduce dose.This conclusion can be by following evidence.
The comparison of content of hesperidin in cholelithiasis treating Danshitongli tablet prepared by test one, distinct methods
1, instrument and reagent cholelithiasis treating Danshitongli tablet of the present invention: press embodiment 1 method preparation, use 1080g crude drug, make 500 through extracting, every heavy 0.3g.Former DanShiTongLi capsules, according to WS-10324(ZD-0324)-2002 standard method preparations, in contrast.Agilent1200 high performance liquid chromatograph; AE240 electronic analytical balance; Hesperidin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (appendix V D of Chinese Pharmacopoeia version in 2010).
Strong to close silica gel be filler by octadecylsilane for chromatographic condition and system suitability; Methanol-water (40: 60) is mobile phase; Detection wavelength is 284nm; Number of theoretical plate calculates and should be not less than 3000 by Hesperidin peak.
It is appropriate that the preparation precision of reference substance solution takes Hesperidin reference substance, adds methanol and make the solution of every 1ml containing 38 μ g, to obtain final product.
Cholelithiasis treating Danshitongli tablet of the present invention is got in the preparation of product need testing solution of the present invention, and porphyrize is got 0.06g,, accurately weighed, put in tool plug conical flask, precision adds Diluted Alcohol 25ml, weighed weight, close plug, supersound process (power 250w, frequency 30kHz) 40 minutes, lets cool, weighed weight, and supply the weight of less loss with Diluted Alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
The DanShiTongLi capsules of this contrast is got in the preparation of reference substance need testing solution, mixes porphyrize, get 0.15g, accurately weighed, put in tool plug conical flask, precision adds Diluted Alcohol 25ml, weighed weight, close plug, supersound process (power 250w, frequency 30kHz) 40 minutes, lets cool, weighed weight, and supply the weight of less loss with Diluted Alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Algoscopy is accurate reference substance solution and the each 10 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and to obtain final product.
3, result
Result shows, in cholelithiasis treating Danshitongli tablet of the present invention, the content of Hesperidin is 6-10mg/ sheet; And the content of Hesperidin is 1.45mg/ grain in former DanShiTongLi capsules, doubly, in the situation that dose reduces, content of hesperidin improves a lot the 4-6 that every content of hesperidin is equivalent to original capsule content.
Above-mentioned research shows, the cholelithiasis treating Danshitongli tablet that adopts preparation method of the present invention to prepare, active constituent content is far away higher than WS-10324(ZD-0324) DanShiTongLi capsules prepared of the method recorded of-2002 standards.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, 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 Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g: become the fine powder of particle size range 5-45 μ m for subsequent use Natrii Sulfas, Alumen micronizing, get Olibanum, Myrrha, be ground into fine powder, 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 25MPa, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 200min, obtains supercritical extract, for subsequent use, Olibanum after extraction, Myrrha residue and other nine tastes medical materials, pulverize, add 70% ethanol of 3.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extract 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, microwave extraction thing mixes and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of Hesperidin is 9.86mg/ sheet.
Embodiment 2
Get Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g: become the fine powder of particle size range 5-45 μ m for subsequent use Natrii Sulfas, Alumen micronizing, get Olibanum, Myrrha, be ground into fine powder, 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 30MPa, temperature 50 C, CO
2flow 3ml/g crude drug min, extraction time 180min, obtains supercritical extract, for subsequent use, Olibanum after extraction, Myrrha residue and other nine tastes medical materials, pulverize, add 70% ethanol of 3.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extract 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, microwave extraction thing mixes and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of Hesperidin is 6.42mg/ sheet.
Embodiment 3
Get Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g: become the fine powder of particle size range 5-45 μ m for subsequent use Natrii Sulfas, Alumen micronizing, get Olibanum, Myrrha, be ground into fine powder, 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 15MPa, 30 ℃ of temperature, CO
2flow 1ml/g crude drug min, extraction time 220min, obtains supercritical extract, for subsequent use, Olibanum after extraction, Myrrha residue and other nine tastes medical materials, pulverize, add 70% ethanol of 3.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 800W, extract 2 times, each 10 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, microwave extraction thing mixes and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of Hesperidin is 8.26mg/ sheet.
Embodiment 4: cholelithiasis treating Danshitongli tablet suppresses the experimentation data of murine myeloma cell SP2/0 cell proliferation
1. experiment material
1.1 experiment cell strains
Murine myeloma cell SP2/0 cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: cholelithiasis treating Danshitongli tablet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg cholelithiasis treating Danshitongli tablet, 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 the use of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
DMEM (Cat.No.12100-061Lot.No.758137 of GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); NaHCO
3(Shanghai hundred million Cat.No.11810-033Lot.No.1088387 of chemical reagent company limited of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); Penicillin G Sodium Salt(AMRESCO company 1); Streptomycin Sulfate (AMRESCO); Dehydrated alcohol (Zibo Ya Dulan Trade Co., Ltd.); MTT (Biosharp lot number: 0793): PBS(laboratory autogamy);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); CO
2incubator (FORMA model: 3111); Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. 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); 1cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) SP2/0 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 concentration of cell suspension 3 × 10
4individual/ml.
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 cholelithiasis treating Danshitongli 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, pats dry gently with absorbent paper, and low-speed oscillation 10min on shaking table, as entered 200 μ l dimethyl sulfoxide, is put in every hole, 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), 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 disposition
Adopt correlation analysis and Student t check in Microsoft Excel 2007 softwares, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, in the time that dosage reaches 5mg/ml, to SP2/0 cell inhibitory effect variant (P<0.05), dosage this difference in the time of 10mg/ml has significance (P<0.01), has utmost point significant difference (P<0.001) in the time that dosage reaches 15-20mg/ml.
Table 1 cholelithiasis treating Danshitongli tablet is on SP2/0 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 9.99±7.02 |
| 2 | 10 | 17.25±9.57* |
| 3 | 15 | 28.46±12.06** |
| 4 | 20 | 38.69±14.06** |
Note: with matched group comparison, * P<0.01; * P<0.001.
5. experiment conclusion
Cholelithiasis treating Danshitongli can suppress SP2/0 cell proliferation, reduces the Growth of Cells number of SP2/0 cell, and this effect is dose dependent.
Claims (9)
1. the preparation method of a cholelithiasis treating Danshitongli tablet, made as crude drug by Herba Lysimachiae 225g, Herba Artemisiae Scopariae 225g, Radix Curcumae 90g, Pericarpium Citri Reticulatae 150g, Radix Scutellariae 45g, Olibanum 35g, Natrii Sulfas 80g, Alumen 35g, Radix Et Rhizoma Rhei 45g, Rhizoma Sparganii 45g, Fructus Gardeniae 45g, Radix Glycyrrhizae 25g, Myrrha 35g, it is characterized in that, described preparation method is made up of the following step: Natrii Sulfas, Alumen micronizing become the fine powder of particle size range 5-45 μ m for subsequent use, get Olibanum, Myrrha, be ground into fine powder, 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 180-220min, obtains supercritical extract, for subsequent use, Olibanum after extraction, Myrrha residue and other nine tastes medical materials, pulverize, add 70% ethanol of 3.6L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, microwave extraction thing mixes and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
2. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
3. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, described CO
2in supercritical extraction, extracting pressure is 25MPa.
4. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, described CO
2in supercritical extraction, extraction temperature is 60 ℃.
5. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
6. according to the preparation method of cholelithiasis treating Danshitongli tablet claimed in claim 1, it is characterized in that described CO
2in supercritical extraction, extraction time is 200min.
7. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, described microwave extracting power is 700W.
8. the preparation method of cholelithiasis treating Danshitongli tablet according to claim 1, is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
9. cholelithiasis treating Danshitongli tablet claimed in claim 1 suppresses the application in murine myeloma cell SP2/0 cell proliferation medicine in preparation.
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| CN104306827A (en) * | 2014-11-13 | 2015-01-28 | 山东中大药业有限公司 | Preparation method of liver benefiting tablet containing nepal dock root and day lily and application of liver benefiting tablet to preparation of drug for restraining multiplication of myeloma cell SP2/0 cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988723A (en) * | 2012-10-08 | 2013-03-27 | 李正梅 | Preparation method and application of yin tonifying and calming tablet |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102988723A (en) * | 2012-10-08 | 2013-03-27 | 李正梅 | Preparation method and application of yin tonifying and calming tablet |
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| 国家药品监督管理局: "胆石通利胶囊", 《国家药品标准（试行）颁布件》 * |
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| CN104306827A (en) * | 2014-11-13 | 2015-01-28 | 山东中大药业有限公司 | Preparation method of liver benefiting tablet containing nepal dock root and day lily and application of liver benefiting tablet to preparation of drug for restraining multiplication of myeloma cell SP2/0 cell |
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