CN103655743B - A kind of preparation method of fukean tablet and the application in preparation suppression mouse lymphoma cell BL4 cell proliferation thereof - Google Patents
A kind of preparation method of fukean tablet and the application in preparation suppression mouse lymphoma cell BL4 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 fukean tablet.The preparation method of fukean tablet of the present invention, be made up as crude drug of Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g, adopt microwave extracting and macroporous resin adsorption to be prepared from, general flavone content is improved a lot.Present invention also offers the application of fukean tablet in preparation suppression mouse lymphoma cell BL4 cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of fukean tablet and suppress the application in mouse lymphoma cell BL4 cell proliferation in preparation.
Background technology
Fukean tablet standard No. WS-10256(ZD-0256)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Be made up as crude drug of Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g, there is clearing away heat-damp and promoting diuresis, convergence, effect of pain relieving.The stomachache caused for acute gastroenteritis, dyspepsia, diarrhoea, vomiting.
In prior art, fukean tablet is not yet had to extract the report adopting microwave technology and macroporous resin adsorption extractive technique in preparation, and decocting in water alcohol extraction, 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.
In prior art, fukean tablet is oral, one time 4,3 times on the one.Fukean tablet dosage is large.The heavy 0.3g of the every sheet of the fukean 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 general flavone content in fukean tablet prepared by test one, distinct methods
L, instrument and reagent fukean tablet of the present invention: by the preparation of embodiment 1 method, use 3330g crude drug, makes 500 through extracting, the heavy 0.3g of every sheet.Former fukean tablet, according to WS-10256(ZD-0256)-2002 standard method preparations, in contrast.Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); METTLER AE240 electronic analytical balance; Control substance of Rutin (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
The preparation precision of reference substance solution takes at 120 DEG C of drying under reduced pressure appropriate to the control substance of Rutin of constant weight, adds 60% ethanol and makes the solution of every 1ml containing 0.1mg, to obtain final product.
The preparation precision of standard curve measures reference substance solution 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, put in 25ml measuring bottle respectively, respectively add 10% sodium nitrite solution 0.3ml, mixing, places 6 minutes, add 10% aluminum nitrate solution 0.3ml, mixing, places 6 minutes, add 4% sodium hydroxide solution 4ml, shake up, add 30% ethanol to scale, shake up, place 15 minutes; With corresponding solution for blank.According to spectrophotography (Chinese Pharmacopoeia version in 2000 annex V B) test, measure trap at 510nm wavelength place, take trap as vertical coordinate, concentration is abscissa, drawing standard curve.
Algoscopy gets this product under weight differential item, removing coating, porphyrize, (fukean tablet prepared of the inventive method gets 0.125g in sampling, former fukean tablet takes 0.2g), accurately weighed, put in round-bottomed flask, add methanol 25ml, shake up, reflux 1 hour, take out, be cooled to room temperature, filter, filter paper adds methanol 25ml again together with residue, reflux 1 hour, take out, cooling, filter, merging filtrate, residue methanol 15ml gradation is washed, washing liquid and filtrate merge, evaporate to dryness, residue adds 60% alcoholic solution makes dissolving, to be transferred in 50ml measuring bottle and to be diluted to scale, shake up.Precision measures 1ml, method under sighting target directrix curve preparation, from " putting respectively in 25ml measuring bottle ", measures trap in accordance with the law, reads the weight of rutin in need testing solution from standard curve, calculates, to obtain final product.
3, result
Result shows, in fukean tablet of the present invention, the content of total flavones counts 80-100mg/ sheet with rutin; And the content of total flavones counts 10.54mg/ sheet with rutin in former fukean tablet, every sheet rutin content is equivalent to the 8-10 of former tablet content doubly, and when dose reduces, general flavone content improves a lot.
Above-mentioned research shows, adopt fukean tablet prepared by preparation method of the present invention, active constituent content is far away higher than WS-10256(ZD-0256) fukean tablet prepared of method recorded of-2002 standards.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of fukean tablet.
Another object of the present invention is to provide a kind of fukean tablet to suppress the application in mouse lymphoma cell BL4 cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of fukean tablet, by Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g makes as crude drug, it is characterized in that, described preparation method is made up of the following step: get upper gomi herbs, pulverize, add 65% ethanol of 10L, 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, 65% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, the mixing of microwave extraction thing is added starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
In the preparation method of above-mentioned fukean tablet, described microwave extracting power is 700W.
In the preparation method of above-mentioned fukean tablet, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned fukean tablet suppresses the application in mouse lymphoma cell BL4 cell proliferation in preparation.
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 Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g, add 65% ethanol of 10L, 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, 65% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, the mixing of microwave extraction thing is added starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
After testing, the content of finished product total flavones counts 98.92mg/ sheet with rutin
Embodiment 2
Get Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g, by gomi herbs, pulverize, add 65% ethanol of 10L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extract 2 times, each 5 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 65% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, the mixing of microwave extraction thing is added starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
After testing, in finished product, the content of total flavones counts 82.16mg/ sheet with rutin.
Embodiment 3
Get Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g, by gomi herbs, pulverize, add 65% ethanol of 10L, drop in microwave extracting apparatus and carry out microwave extracting, 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, 65% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, the mixing of microwave extraction thing is added starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
After testing, in finished product, in finished product, the content of total flavones counts 90.44mg/ sheet with rutin.
Embodiment 4: fukean tablet suppresses the experimentation data of mouse lymphoma cell BL4 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse lymphoma cell BL4 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: fukean tablet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg fukean 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); 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 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) BL4 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 fukean tablet solution, continue to cultivate 24h.
4) add 20 μ l MTT 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 Microsoft Excel2007 software and Student t to check, 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 BL4 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 fukean tablet is to BL4 cell inhibitory effect influence research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 9.77±9.04 |
| 2 | 10 | 17.61±12.71* |
| 3 | 15 | 33.85±15.37** |
| 4 | 20 | 40.54±18.63** |
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Fukean tablet can suppress BL4 cell proliferation, and reduce the Growth of Cells number of BL4 cell, this effect is dose dependent.
Claims (3)
1. the application of fukean tablet in preparation suppression mouse lymphoma cell BL4 cell proliferation, it is characterized in that, described fukean tablet is by Herba Oxalidis strictae 1000g, Herb Polygoni Chinensis 1000g, Cortex Ilicis Rotundae 670g, Herba Plantaginis 330g, Pericarpium Granati 330g makes as crude drug, the preparation method of described fukean tablet is made up of the following step: get upper gomi herbs, pulverize, add 65% ethanol of 10L, 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, 65% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, the mixing of microwave extraction thing is added starch, 70% ethanol granule, dry, tabletting, make 500, the heavy 0.3g of every sheet.
2. the fukean tablet according to claim l suppresses the application in mouse lymphoma cell BL4 cell proliferation in preparation, and it is characterized in that, described microwave extracting power is 700W.
3. the fukean tablet according to claim l suppresses the application in mouse lymphoma cell BL4 cell proliferation in preparation, and it is characterized in that, each extraction time of described microwave extracting is 8 minutes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1887315A (en) * | 2006-05-24 | 2007-01-03 | 张嵩 | Fukean disperser tablet and its prepn |
| CN102127142A (en) * | 2010-12-28 | 2011-07-20 | 赵全成 | Ilicis routundae cortex derivants and application thereof in preparing medicament capable of resisting tumors |
| CN102824409A (en) * | 2012-09-24 | 2012-12-19 | 南京正亮医药科技有限公司 | Preparation method of liver protection tablets |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1887315A (en) * | 2006-05-24 | 2007-01-03 | 张嵩 | Fukean disperser tablet and its prepn |
| CN102127142A (en) * | 2010-12-28 | 2011-07-20 | 赵全成 | Ilicis routundae cortex derivants and application thereof in preparing medicament capable of resisting tumors |
| CN102824409A (en) * | 2012-09-24 | 2012-12-19 | 南京正亮医药科技有限公司 | Preparation method of liver protection tablets |
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