CN103768262A - Preparation method of Qingmei cold tablet and application of Qingmei cold tablet in drugs for inhibiting myeloma cell FO from cell proliferation - Google Patents
Preparation method of Qingmei cold tablet and application of Qingmei cold tablet in drugs for inhibiting myeloma cell FO 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 Qingmei cold tablet. The preparation method comprises the following steps: by using 200g of southernwood, 267g of finger citron, 134g of elsholtzia, 200g of dicliptera, 134g of licorice, 134g of cat's-foot and 267g of holly root as active pharmaceutical ingredients, carrying out supercritical extraction and microwave extraction so that the glycyrrhizic acid content is greatly enhanced. The invention also provides application of the Qingmei cold tablet in preparing drugs for inhibiting mouse myeloma cell FO 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 Armeniaca mume Sieb. common cold tablet and the application in inhibition murine myeloma cell FO cell proliferation medicine thereof.
Background technology
Armeniaca mume Sieb. cold granules standard No. WS-11318 (ZD-1318)-2002, is recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Made as crude drug by Herba Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, there is refrigerant antipyretic effect.For anemopyretic cold, headache, cough, nasal obstruction.
In prior art, not yet have Armeniaca mume Sieb. cold granules at the report that adopts supercritical and microwave technology aspect extraction preparation, and volatile oil extracts, the method that decocting boils, technique is coarse, backward, and impurity is many, cause patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of Armeniaca mume Sieb. common cold tablet.
Another object of the present invention is to provide a kind of Armeniaca mume Sieb. common cold tablet to suppress the application in murine myeloma cell FO cell proliferation medicine in preparation.
The object of the invention is to realize by following scheme:
A kind of preparation method of Armeniaca mume Sieb. common cold tablet, made as crude drug by Herba Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, described preparation method is made up of the following step: get Herba Artemisiae Annuae, Herba Moslae, 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 l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 150, every heavy 0.3g.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold 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 Armeniaca mume Sieb. common cold tablet, described CO
2in supercritical extraction, extracting pressure is 25MPa.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO
2in supercritical extraction, extraction temperature is 60 ℃.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described microwave extracting power is 700W.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned Armeniaca mume Sieb. common cold tablet suppresses the application in murine myeloma cell FO cell proliferation medicine in preparation.
In prior art, each 1 bag of Armeniaca mume Sieb. cold granules, every bag of 15g, 2-3 time on the one.Armeniaca mume Sieb. cold granules dosage is large, and granule preparation need to add a large amount of sugar, and diabetics is not suitable for, and the words taste of not sugaring is not good, and patient is not easy under clothes, and compliance is poor.The every heavy 0.3g of Armeniaca mume Sieb. common cold tablet that adopts the inventive method to be prepared into only needs 2 at every turn, within 1st, takes 2-3 time.Under the condition with more active component, greatly reduce dose.This conclusion can be by following evidence.And be prepared into tablet, patient swallows with water, dosage is little and without bitterness, patient is easy to accept.
The comparison of glycyrrhizic acid content in Armeniaca mume Sieb. common cold tablet prepared by test one, distinct methods
L, instrument and reagent Armeniaca mume Sieb. common cold tablet of the present invention: press embodiment 1 method preparation, use 1510g crude drug, make 150 through extracting, every heavy 0.3g.Former Armeniaca mume Sieb. cold granules, prepares according to WS-11318 (ZD-1318)-2002 standard method.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Glycyrrhizic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Methanol-0.2mol/L Spirit of Mindererus .-glacial acetic acid (67: 33: 1) is mobile phase; Detection wavelength is 250nm.Number of theoretical plate calculates and should be not less than 3000 by glycyrrhizic acid peak.
It is appropriate that the preparation precision of reference substance solution takes monoammonium glycyrrhizinate reference substance, adds 90% methanol and make the solution of every 1ml containing 0.4mg, to obtain final product.
Armeniaca mume Sieb. common cold tablet of the present invention is got in the preparation of need testing solution, and porphyrize, gets 0.08g, accurately weighed, to put in tool plug conical flask, precision adds 90% methanol 10ml, close plug, weighed weight, supersound process 30 minutes, let cool, more weighed weight, supply the weight of less loss with 90% methanol, centrifugal, (0.45 μ m) filters microporous filter membrane for supernatant, gets subsequent filtrate, to obtain final product.
The Armeniaca mume Sieb. cold granules of this contrast is got in the preparation of reference product need testing solution, and porphyrize, mixes, get 2g, accurately weighed, put in tool plug conical flask, precision adds 90% methanol 10ml, close plug, weighed weight, supersound process 30 minutes, lets cool, more weighed weight, supply the weight of less loss with 90% methanol, centrifugal, (0.45 μ m) filters microporous filter membrane for supernatant, get subsequent filtrate, to obtain final product.
Algoscopy is accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and to obtain final product.3, result
Result shows, in Armeniaca mume Sieb. common cold tablet of the present invention, the content of glycyrrhizic acid is 35-70mg/ sheet; And the content of glycyrrhizic acid is 14.5mg/ bag in former Armeniaca mume Sieb. cold granules, be 5-10 times of former granule content each serving the glycyrrhizic acid content of 2 of consumptions, in the situation that dose reduces, glycyrrhizic acid content improves a lot.
Above-mentioned research shows, the Armeniaca mume Sieb. common cold tablet that adopts preparation method of the present invention to prepare, Armeniaca mume Sieb. cold granules prepared by the method that active constituent content is recorded higher than WS-11318 (ZD-1318)-2002 standard far away.
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 Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, Herba Artemisiae Annuae, Herba Moslae are joined to 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; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 150, every heavy 0.3g.
After testing, in finished product, the content of glycyrrhizic acid is 69.8mg/ sheet.
Embodiment 2
Get Herba Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, Herba Artemisiae Annuae, Herba Moslae are joined to 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; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 5 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 150, every heavy 0.3g.
After testing, in finished product, the content of glycyrrhizic acid is 56.2mg/ sheet.
Embodiment 3
Get Herba Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, Herba Artemisiae Annuae, Herba Moslae are joined to 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; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 800W, extracts 2 times, each 10 minutes, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 150, every heavy 0.3g.
After testing, in finished product, the content of glycyrrhizic acid is 36.9mg/ sheet.
Embodiment 4: Armeniaca mume Sieb. common cold tablet suppresses the experimentation data of murine myeloma cell FO cell proliferation
1. experiment material
1.1 experiment cell strains
Murine myeloma cell FO cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Armeniaca mume Sieb. common cold tablet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg Armeniaca mume Sieb. common cold 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); NaHC0
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); C0
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) FO cell uses DMEM+10%FBS in 37 ℃, 5%C0
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%C0
2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add Armeniaca mume Sieb. common cold 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 Excel2007 software, 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 FO 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 Armeniaca mume Sieb. common cold tablet is on FO cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 8.86±7.21 |
| 2 | 10 | 18.23±12.12* |
| 3 | 15 | 35.16±17.94** |
| 4 | 20 | 48.65±19.841** |
Note: with matched group comparison, * P<O.01; * P<0.001.
5. experiment conclusion
Armeniaca mume Sieb. common cold tablet of the present invention can suppress FO cell proliferation, reduces the Growth of Cells number of FO cell, and this effect is dose dependent.
Claims (9)
1. the preparation method of an Armeniaca mume Sieb. common cold tablet, made as crude drug by Herba Artemisiae Annuae 200g, Folium vilicis Negundo 267g, Herba Moslae 134g, Herba Diclipterae Chinensis 200g, Radix Glycyrrhizae 134g, Herba Centellae 134g, Flos Ilicis Asprellae 267g, it is characterized in that, described preparation method is made up of the following step: get Herba Artemisiae Annuae, Herba Moslae, 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 l-3ml/g crude drug min, extraction time 180-220min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collects 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 150, every heavy 0.3g.
2. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
3. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, extracting pressure is 25MPa.
4. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction temperature is 60 ℃.
5. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
6. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction time is 200min.
7. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that, described microwave extracting power is 700W.
8. according to the preparation method of the Armeniaca mume Sieb. common cold tablet described in claim l, it is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
9. the Armeniaca mume Sieb. common cold tablet described in claim l suppresses the application in murine myeloma cell FO cell proliferation medicine in preparation.
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| CN104288653A (en) * | 2014-10-16 | 2015-01-21 | 济南新起点医药科技有限公司 | Preparation method of fomes officinalis cough and asthma treating tablets and application of fomes officinalis cough and asthma treating tablets in preparation of medicine for inhibiting cell proliferation of mouse myeloma cell FO |
| CN104398928A (en) * | 2014-11-13 | 2015-03-11 | 山东中大药业有限公司 | Preparation method of rumex japonicus houtt-tawny daylily liver-tonifying tablet and application of rumex japonicus houtt-tawny daylily liver-tonifying tablet in preparation of drugs for inhibition of cell proliferation of myeloma cell FO |
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