CN103751701B - A kind of preparation method of cholelithiasis treating Danshitongli tablet and the application in preparation suppression lymphoma cell YAC-1 cell proliferation thereof - Google Patents
A kind of preparation method of cholelithiasis treating Danshitongli tablet and the application in preparation suppression lymphoma cell YAC-1 cell proliferation thereof Download PDFInfo
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Abstract
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application of cholelithiasis treating Danshitongli tablet.The preparation method of cholelithiasis treating Danshitongli tablet of the present invention, 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, adopt micronizing, supercritical extraction, microwave extracting and macroporous resin adsorption to be prepared from, content of hesperidin is improved a lot.Present invention also offers the application of cholelithiasis treating Danshitongli tablet in preparation suppression mouse lymphoma cell YAC-1 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 mouse lymphoma cell YAC-1 cell proliferation 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.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, there is clearing heat secreting bile, blood stasis dispelling calculus removing effect.For the acute and chronic cholecystitis caused by dampness-heat in the liver and gallbladder, cholelithiasis etc.
In prior art, DanShiTongLi capsules is not yet had to adopt micronizing, CO in extraction preparation
2the report that supercritical extraction, microwave technology and macroporous adsorption resin technology extract, and Chinese medicine is directly beaten, and powder is got, the method for soak by water, 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 mouse lymphoma cell YAC-1 cell proliferation in preparation.
The object of the invention is by following scheme realize:
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 μm 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 DEG C, 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 DiaionHP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, the mixing of microwave extraction thing adds starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
In the preparation method of above-mentioned cholelithiasis treating Danshitongli tablet, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
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 DEG C.
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, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned cholelithiasis treating Danshitongli tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation in preparation.
In prior art, DanShiTongLi capsules is oral, one time 5,3 times on the one.DanShiTongLi capsules dosage is large.The heavy 0.3g of the every sheet of the cholelithiasis treating Danshitongli tablet adopting the inventive method to be prepared into, only needs 2 at every turn, within 1st, takes 3 times.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.
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: by the preparation of embodiment 1 method, use 1080g crude drug, makes 500 through extracting, the heavy 0.3g of every sheet.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 (Chinese Pharmacopoeia version in 2010 annex VD).
Chromatographic condition and system suitability octadecylsilane are good for and are closed silica gel is filler; Methanol-water (40: 60) is mobile phase; Determined wavelength is 284nm; Number of theoretical plate calculates should be not less than 3000 by Hesperidin peak.
The preparation precision of reference substance solution takes Hesperidin reference substance in right amount, adds methanol and makes the solution of every 1ml containing 38 μ g, to obtain final product.
The preparation of product need testing solution of the present invention gets cholelithiasis treating Danshitongli tablet of the present invention, porphyrize, gets 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 the weight of supplying 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, mixing, 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 the weight of supplying less loss with Diluted Alcohol, shake up, filter, get subsequent filtrate, to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, 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, every sheet content of hesperidin is equivalent to the 4-6 of original capsule content doubly, and when dose reduces, content of hesperidin improves a lot.
Above-mentioned research shows, adopt cholelithiasis treating Danshitongli tablet prepared by preparation method of the present invention, active constituent content is far away higher than WS-10324(ZD-0324) DanShiTongLi capsules prepared of method recorded of-2002 standards.
Detailed description of the invention
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 that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized 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 μm 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, temperature 40 DEG C, 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 DiaionHP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, the mixing of microwave extraction thing adds starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
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 μm 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 DiaionHP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, the mixing of microwave extraction thing adds starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
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 μm 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, temperature 30 DEG C, 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 DiaionHP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, the mixing of microwave extraction thing adds starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
After testing, in finished product, the content of Hesperidin is 8.26mg/ sheet.
Embodiment 4: cholelithiasis treating Danshitongli tablet suppresses the experimentation data of mouse lymphoma cell YAC-1 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse lymphoma cell YAC-1 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: cholelithiasis treating Danshitongli tablet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg cholelithiasis treating Danshitongli tablet, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHCO
3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); PenicillinGSodiumSalt(AMRESCO company 1); StreptomycinSulfate (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 Inc. of Su Jing group moulding number: 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 (the accurate experimental facilities company in Shanghai 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 μm of 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) YAC-1 cell DMEM+10%FBS is in 37 DEG C, 5%CO
2carry out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 10
4individual/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, culture plate put into cell culture incubator (37 DEG C, 5%CO
2) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add cholelithiasis treating Danshitongli tablet solution, continue to cultivate 24h.
4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole, as entered 200 μ l dimethyl sulfoxide, is put low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.
7) result represents with the suppression ratio of medicine to cell: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repetition 3 times.
3. statistical disposition
Adopt the correlation analysis in MicrosoftExcel2007 software and Studentt inspection, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to YAC-1 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has pole significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 cholelithiasis treating Danshitongli tablet is to YAC-1 cell inhibitory effect influence research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 8.42±7.03 |
| 2 | 10 | 15.98±8.69* |
| 3 | 15 | 27.03±10.77** |
| 4 | 20 | 36.56±12.64** |
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Cholelithiasis treating Danshitongli tablet can suppress YAC-1 cell proliferation, and reduce the Growth of Cells number of YAC-1 cell, this effect is dose dependent.
Claims (2)
1. the application of cholelithiasis treating Danshitongli tablet in preparation suppression lymphoma cell YAC-1 cell proliferation, the preparation method of described cholelithiasis treating Danshitongli tablet is as follows: 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, 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 μm 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 5%, extracting pressure 25MPa, temperature 30-50 DEG C, 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 5-10 minute, combining extraction liquid, concentrated, be added on DiaionHP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, by the Natrii Sulfas after micronizing, Alumen, supercritical extract, the mixing of microwave extraction thing adds starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
2. the cholelithiasis treating Danshitongli tablet according to claim l suppresses the application in lymphoma cell YAC-1 cell proliferation in preparation, and it is characterized in that, in the preparation method of described cholelithiasis treating Danshitongli tablet, each extraction time of described microwave extracting is 8 minutes.
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| 胆石通利胶囊;国家药品监督管理局;《国家药品标准（试行）颁布件》;20021116;"处方"和"制法" * |
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