CN102988550B - Preparation method and application of qinlian tablet - Google Patents
Preparation method and application of qinlian tablet Download PDFInfo
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- CN102988550B CN102988550B CN2012103883322A CN201210388332A CN102988550B CN 102988550 B CN102988550 B CN 102988550B CN 2012103883322 A CN2012103883322 A CN 2012103883322A CN 201210388332 A CN201210388332 A CN 201210388332A CN 102988550 B CN102988550 B CN 102988550B
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Abstract
The invention provides a preparation method of a qinlian tablet. The qinlian tablet is prepared by using 250g of baical skullcap roots, 250g of fructus forsythia, 100g of coptis chinensis, 400g of amur corktree bark, 250g of red paeony roots and 100g of licorice roots as bulk drugs and adopting supercritical extraction and microwave-assisted extraction modes. By adopting the method, the content of baicalein is greatly improved; and the invention also provides application of the qinlian tablet in preparation of drugs for inhibiting cell proliferation of mice myeloma SP2/0.
Description
Technical field
The present invention relates to the Chinese medicine preparation technical field, be specifically related to preparation method and the application in flakes of a kind of a kind of reed mentioned in ancient books.
Background technology
A kind of reed mentioned in ancient books is recorded in Ministry of Public Health standard WS3-B-0551-91 in flakes, is made energy heat-clearing and toxic substances removing, reducing swelling and alleviating pain as crude drug by Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g.For the internal organs intrinsic heat, cause: the headache conjunctival congestion, mouth and nose are given birth to skin ulcer, hematodiarrhoea stomachache, damp-heat vaginal discharge, furuncle swells and ache.
In prior art, not yet there is a kind of reed mentioned in ancient books to adopt the report of supercritical and microwave technology extracting aspect preparation in flakes, 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 a kind of reed mentioned in ancient books preparation method in flakes.
Another object of the present invention is to provide a kind of a kind of reed mentioned in ancient books to suppress the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of a kind of reed mentioned in ancient books preparation method in flakes, by Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g, as crude drug, made, described method is comprised of the following step: get Fructus Forsythiae, join in CO2 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, obtain 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, extract 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
Above-mentioned a kind of a kind of reed mentioned in ancient books preparation method in flakes, described CO
2the percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
Above-mentioned a kind of a kind of reed mentioned in ancient books preparation method in flakes, described microwave extracting power 500W extracts 6 minutes at every turn.
Above-mentioned a kind of a kind of reed mentioned in ancient books preparation method in flakes, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned a kind of reed mentioned in ancient books suppresses the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes.
In prior art, a kind of reed mentioned in ancient books is every 0.55g in flakes, and each 4,2-3 time on the one, a kind of reed mentioned in ancient books that adopts the present invention to be prepared into is every 0.5g in flakes, only needs 2 at every turn, within 1st, takes 2 times, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
A kind of reed mentioned in ancient books prepared by test one, distinct methods is the comparison of middle baicalin content in flakes
1, instrument and reagent a kind of reed mentioned in ancient books of the present invention in flakes: press embodiment 3 method preparations, use the 1350g crude drug, through extraction, make 1000, every heavy 0.5g.Former a kind of reed mentioned in ancient books in flakes, by the preparation of ministry standard method, is used the 1350g crude drug, through extraction, makes 1175, every heavy 0.55g.The Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Baicalin 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; The detection wavelength is 280nm.Number of theoretical plate is pressed the baicalin 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 baicalin reference substance of 4 hours appropriate, adds methanol and makes the solution of every 1ml containing 18 μ g, obtains.
The preparation of need testing solution: get a kind of reed mentioned in ancient books of the present invention in flakes with former a kind of reed mentioned in ancient books in flakes, porphyrize, mix, 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 the injection liquid chromatography, measure, and obtains.
3, result
Result shows, a kind of reed mentioned in ancient books of the present invention in flakes in the content of baicalin be the 1.84mg/ sheet; And former a kind of reed mentioned in ancient books in flakes in the content of baicalin be the 0.35mg/ sheet, in the situation that dose reduces, baicalin content improves a lot.
Above-mentioned research shows, in flakes, active constituent content a kind of reed mentioned in ancient books standby higher than the ministry standard legal system in flakes for a kind of reed mentioned in ancient books that adopts the present invention to prepare.
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 realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g, by Fructus Forsythiae, join in CO2 supercritical extraction device, ethanol is as entrainer, and 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, obtain 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, extract 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is the 1.95mg/ sheet.
Embodiment 2
Get Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g, by Fructus Forsythiae, 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, obtain 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, extract 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is the 1.79mg/ sheet.
Embodiment 3
Get Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g, by Fructus Forsythiae, 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, obtain 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, extract 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
After testing, in finished product, the content of baicalin is the 1.84mg/ sheet.
Embodiment 4: a kind of reed mentioned in ancient books suppresses the experimentation data of SP2/0 cell proliferation in flakes
1 experiment material
1.1 experiment cell strain
Mouse myeloma SP2/0 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: a kind of reed mentioned in ancient books of the present invention in flakes: press embodiment 3 methods preparations.
The medicinal liquid liquid storage: take 100mg a kind of reed mentioned in ancient books in flakes, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe packing, and-20 ℃ of storages, 0.2 μ m filter filters the use of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061Lot.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.11810-033Lot.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); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX190); 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) the SP2/0 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, discard 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 the piping and druming cell, it is proceeded in centrifuge tube, the centrifugal 5min of 1000rpm, adjust 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 a kind of reed mentioned in ancient books solution in flakes, 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, the 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.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), set 6 multiple holes for every group.
7) with medicine, the suppression ratio to cell means result:
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 mean with mean ± S.D..
4 experimental results
Statistical result showed after the mtt assay experiment, with matched group, compare, when dosage reaches 5mg/ml, to SP2/0 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 a kind of reed mentioned in ancient books is in flakes on SP2/0 cell inhibitory effect impact research
Annotate: compare * P<0.01 with matched group; * P<0.001
5 experiment conclusion
A kind of reed mentioned in ancient books can suppress the SP2/0 cell proliferation in flakes, reduces the Growth of Cells number of SP2/0 cell, and this effect is dose dependent.
Claims (4)
1. a kind of reed mentioned in ancient books suppresses the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes, it is characterized in that a kind of reed mentioned in ancient books preparation method in flakes is as follows, by Radix Scutellariae 250g, Fructus Forsythiae 250g, Rhizoma Coptidis 100g, Cortex Phellodendri 400g, Radix Paeoniae Rubra 250g, Radix Glycyrrhizae 100g, as crude drug, made, described method is comprised of the following step: get Fructus Forsythiae, 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, obtain 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, extract 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain the microwave extraction thing, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, drying, tabletting, make 1000, every heavy 0.5g.
2. a kind of a kind of reed mentioned in ancient books suppresses the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes according to claim 1, it is characterized in that CO described in a kind of reed mentioned in ancient books preparation method in flakes
2the percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
3. a kind of a kind of reed mentioned in ancient books suppresses the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes according to claim 1, it is characterized in that microwave extracting power 500W described in a kind of reed mentioned in ancient books preparation method in flakes, extracts 6 minutes at every turn.
4. a kind of a kind of reed mentioned in ancient books suppresses the application in mouse myeloma SP2/0 cell proliferation medicine in preparation in flakes according to claim 1, it is characterized in that CO described in a kind of reed mentioned in ancient books preparation method in flakes
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103883322A CN102988550B (en) | 2012-10-15 | 2012-10-15 | Preparation method and application of qinlian tablet |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103883322A CN102988550B (en) | 2012-10-15 | 2012-10-15 | Preparation method and application of qinlian tablet |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102988550A CN102988550A (en) | 2013-03-27 |
| CN102988550B true CN102988550B (en) | 2013-12-11 |
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| CN104971124B (en) * | 2014-04-10 | 2019-05-24 | 通威股份有限公司 | A kind of new application of pharmaceutical composition |
| CN106581309A (en) * | 2016-11-30 | 2017-04-26 | 四川旭华制药有限公司 | Traditional Chinese medicine for clearing away heat and toxic materials |
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