CN102988612A - Preparation method and application of tablet for clearing away heat - Google Patents
Preparation method and application of tablet for clearing away heat Download PDFInfo
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- CN102988612A CN102988612A CN2012103900737A CN201210390073A CN102988612A CN 102988612 A CN102988612 A CN 102988612A CN 2012103900737 A CN2012103900737 A CN 2012103900737A CN 201210390073 A CN201210390073 A CN 201210390073A CN 102988612 A CN102988612 A CN 102988612A
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Abstract
The invention provides a preparation method of tablet for clearing away heat. The tablet is prepared by using 400g of indigowoad leaves, 100g of rhubarb, 50g of gypsum and 0.65g of menthol as bulk drugs and adopting supercritical extraction and microwave-assisted extraction modes. By adopting the method, the content of rheum emodin is greatly improved; and the invention also provides application of the tablet in preparation of drugs for inhibiting cell proliferation of rat neurospongioma cells C6.
Description
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 Qinghuo tablet.
Background technology
Qinghuo tablet is recorded in Ministry of Public Health standard WS3-B-0414-90, is made energy clearing away heat-fire, relieving constipation as crude drug by Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g.Be used for laryngopharynx swelling and pain, toothache, the vertigo, mouth and nose are given birth to skin ulcer, wind-fire conjunctival congestion, constipation.
In the prior art, not yet there is Qinghuo 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 Qinghuo tablet.
Another object of the present invention is to provide the application of a kind of Qinghuo tablet in preparation inhibition rat neurogliocytoma cells C6 cell proliferation medicine.
Technical scheme: the objective of the invention is to realize by following scheme:
A kind of preparation method of Qinghuo tablet, made as crude drug by Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g, described method is comprised of the following step: get Folium Isatidis, Mentholum, join in the 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 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 0.5g.
The preparation method of above-mentioned a kind of Qinghuo 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 Qinghuo tablet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of Qinghuo 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 Qinghuo tablet in preparation inhibition C6 cell proliferation medicine.
In the prior art, every 0.5g of Qinghuo tablet, each 6,2 times on the one, the every 0.5g of Qinghuo tablet that adopts the present invention to be prepared into only needs 3 at every turn, takes 2 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 emodin content in the Qinghuo tablet of test one, distinct methods preparation
1, instrument and reagent Qinghuo tablet of the present invention: press the preparation of embodiment 3 methods, use the 550.65g crude drug, make 1000 through extraction, every heavy 0.5g.Former Qinghuo tablet by the preparation of ministry standard method, uses the 550.65g crude drug, makes 1000 through extraction, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; The METTLERAE240 electronic analytical balance; Emodin 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 emodin peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours emodin 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 Qinghuo tablet of the present invention and former Qinghuo 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, the content of emodin is the 1.34mg/ sheet in the Qinghuo tablet of the present invention; And the content of emodin is the 0.26mg/ sheet in the former Qinghuo tablet, in the situation that dose reduces, emodin content improves a lot.
Above-mentioned studies show that, the Qinghuo tablet that adopts the present invention to prepare, active constituent content is higher than the standby Qinghuo 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 Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g, with Folium Isatidis, Mentholum, join CO
2In the 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 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 0.5g.
After testing, the content of emodin is the 1.48mg/ sheet in the finished product.
Embodiment 2
Get Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g, with Folium Isatidis, Mentholum, 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 0.5g.
After testing, the content of emodin is the 1.33mg/ sheet in the finished product.
Embodiment 3
Get Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g, with Folium Isatidis, Mentholum, 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 0.5g.
After testing, the content of emodin is the 1.34mg/ sheet in the finished product.
Embodiment 4: Qinghuo tablet suppresses the experimentation data of C6 cell proliferation
1 experiment material
1.1 experiment cell strain
Rat neurogliocytoma cells (C6), Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Qinghuo tablet of the present invention: press the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing the 100mg Qinghuo tablet, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ l doff 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.12100-061Lot.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); 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 C6 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 Qinghuo 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 C6 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 Qinghuo tablet is on C6 cell inhibitory effect impact research
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
Qinghuo tablet can suppress the C6 cell proliferation, reduces the Growth of Cells number of C6 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of a Qinghuo tablet, made as crude drug by Folium Isatidis 400g, Radix Et Rhizoma Rhei 100g, Gypsum Fibrosum 50g, Mentholum 0.65g, it is characterized in that described method is comprised of the following step: get Folium Isatidis, Mentholum, join in the 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 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 0.5g.
2. the preparation method of described a kind of Qinghuo 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 Qinghuo 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 Qinghuo 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 Qinghuo tablet in preparation inhibition rat neurogliocytoma cells C6 cell proliferation medicine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104306830A (en) * | 2014-11-13 | 2015-01-28 | 山东中大药业有限公司 | Preparing method of rhubarb and day lily liver tonifying tablet and application thereof in preparation of drugs for inhibiting cell proliferation of myeloma cells P3X63Ag8 |
| CN104306822A (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 liver tumor cell H22 cell |
| CN104434827A (en) * | 2014-11-13 | 2015-03-25 | 山东中大药业有限公司 | Preparation method of rumex nepalensis and hemerocallis tablet for benefiting liver and application of rumex nepalensis and hemerocallis tablet to preparation of drug for inhibiting proliferation of teratocarcinoma cell P19 |
| CN105748615A (en) * | 2016-02-28 | 2016-07-13 | 南京正亮医药科技有限公司 | Method for preparing tablet containing rheum officinale, berberine hydrochloride and scutellaria baicalensis and application thereof |
| WO2021253803A1 (en) * | 2020-06-18 | 2021-12-23 | 海南葫芦娃药业集团股份有限公司 | Preparation method for fire-purging tablet |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104306830A (en) * | 2014-11-13 | 2015-01-28 | 山东中大药业有限公司 | Preparing method of rhubarb and day lily liver tonifying tablet and application thereof in preparation of drugs for inhibiting cell proliferation of myeloma cells P3X63Ag8 |
| CN104306822A (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 liver tumor cell H22 cell |
| CN104434827A (en) * | 2014-11-13 | 2015-03-25 | 山东中大药业有限公司 | Preparation method of rumex nepalensis and hemerocallis tablet for benefiting liver and application of rumex nepalensis and hemerocallis tablet to preparation of drug for inhibiting proliferation of teratocarcinoma cell P19 |
| CN105748615A (en) * | 2016-02-28 | 2016-07-13 | 南京正亮医药科技有限公司 | Method for preparing tablet containing rheum officinale, berberine hydrochloride and scutellaria baicalensis and application thereof |
| WO2021253803A1 (en) * | 2020-06-18 | 2021-12-23 | 海南葫芦娃药业集团股份有限公司 | Preparation method for fire-purging tablet |
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| CN102988612B (en) | 2014-05-07 |
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