The preparation method of a kind of Armeniaca mume Sieb. common cold tablet and the application in suppressing stomach cancer cell MFC cell proliferation thereof
Grow the application in medicine
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the preparation method of a kind of Armeniaca mume Sieb. common cold tablet and the application in suppressing Mouse Gastric Cancer cell MFC cell proliferation thereof.
Background technology
Armeniaca mume Sieb. cold granules standard No. WS-11318 (ZD-1318)-2002, is recorded in country's standard for traditional Chinese medicines compilation internal medicine taste fascicle.It is made up as crude drug of 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 there is Armeniaca mume Sieb. cold granules extracting the report adopting supercritical and microwave technology in preparation, and volatile oil is drawn, the method of soak by water, technique is coarse, backward, and impurity is many, cause that patient's consumption is excessive, it has not been convenient 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 preparation method that the invention provides a kind of Armeniaca mume Sieb. common cold tablet.
Further object is that and provide a kind of Armeniaca mume Sieb. common cold tablet to suppress the application in Mouse Gastric Cancer cell MFC cell proliferation in preparation.
It is an object of the invention to by following scheme realization:
A kind of preparation method of Armeniaca mume Sieb. common cold tablet, it is made up as crude drug of 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: takes Herba Artemisiae Annuae, Herba Moslae, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 180-220min, obtain supercritical extract, standby;Take all the other Chinese medicines, pulverize, add 50% ethanol of 5L, put into and microwave extracting apparatus carries out microwave extracting, extract power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentration, it is added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain microwave extraction thing, standby;Above-mentioned supercritical extract and microwave extraction thing are mixed, adds starch, 70% ethanol granule, dry, tabletting, make 150, every tablet weight 0.3g.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO2In supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO2In supercritical extraction, extracting pressure is 25MPa.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO2In supercritical extraction, extraction temperature is 60 DEG C.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO2CO in supercritical extraction2Flow 2ml/g crude drug min.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO2In 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, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned Armeniaca mume Sieb. common cold tablet suppresses the application in Mouse Gastric Cancer cell MFC cell proliferation 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 big, and granule preparation needs to add substantial amounts 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 tablet weight 0.3g of Armeniaca mume Sieb. common cold tablet adopting the inventive method to prepare, only needs 2 every time, within 1st, takes 2-3 time.Dose is greatly reduced when having more active component.This conclusion can pass through following it have been experienced that.And preparing into tablet, patient swallows with water, and dosage is little and without bitterness, and patient is prone 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: prepare by embodiment 1 method, uses 1510g crude drug, extracted makes 150, every tablet weight 0.3g.Former Armeniaca mume Sieb. cold granules, prepares according to WS-11318 (ZD-1318)-2002 standard method.Agilent1200 high performance liquid chromatograph;METTLERAE240 electronic analytical balance;Glycyrrhizic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia one annex VI D of 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 by glycyrrhizic acid peak should be not less than 3000.
It is appropriate that the preparation precision of reference substance solution weighs monoammonium glycyrrhizinate reference substance, adds 90% methanol and makes every 1ml solution containing 0.4mg, to obtain final product.
The preparation of need testing solution takes the Armeniaca mume Sieb. common cold tablet of the present invention, finely ground, takes 0.08g, accurately weighed, put in tool plug conical flask, accurate addition 90% methanol 10ml, close plug, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with 90% methanol, centrifugal, supernatant microporous filter membrane (0.45 μm) filters, and takes subsequent filtrate, to obtain final product.
The preparation of reference product need testing solution takes the Armeniaca mume Sieb. cold granules of this comparison, finely ground, mixing, take 2g, accurately weighed, put in tool plug conical flask, accurate addition 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, supernatant microporous filter membrane (0.45 μm) filters, take subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 20 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.3, result
It is shown that the content of glycyrrhizic acid is 35-70mg/ sheet in Armeniaca mume Sieb. common cold tablet of the present invention;And the content of glycyrrhizic acid is 14.5mg/ bag in former Armeniaca mume Sieb. cold granules, each serving 5-10 times that glycyrrhizic acid content is former granule content of consumption 2, when dose reduces, glycyrrhizic acid content improves a lot.
The studies above shows, adopts Armeniaca mume Sieb. common cold tablet prepared by preparation method of the present invention, and active constituent content is significantly larger than Armeniaca mume Sieb. cold granules prepared by the method for WS-11318 (ZD-1318)-2002 standard record.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art knows more about the present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, and all technology realized based on foregoing of the present invention belong to the scope of the present invention.
Embodiment 1
Take 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 CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 200min, obtain supercritical extract, standby;Take all the other Chinese medicines, pulverize, add 50% ethanol of 5L, put into and microwave extracting apparatus carries out microwave extracting, extract power 700W, extract 2 times, each 8 minutes, combining extraction liquid, concentration, it is added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain microwave extraction thing, standby;Above-mentioned supercritical extract and microwave extraction thing are mixed, adds starch, 70% ethanol granule, dry, tabletting, make 150, every tablet weight 0.3g.
After testing, in finished product, the content of glycyrrhizic acid is 69.8mg/ sheet.
Embodiment 2
Take 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 CO2In supercritical extraction device, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature 50 C, CO2Flow 3ml/g crude drug min, extraction time 180min, obtain supercritical extract, standby;Take all the other Chinese medicines, pulverize, add 50% ethanol of 5L, put into and microwave extracting apparatus carries out microwave extracting, extract power 600W, extract 2 times, each 5 minutes, combining extraction liquid, concentration, it is added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain microwave extraction thing, standby;Above-mentioned supercritical extract and microwave extraction thing are mixed, adds starch, 70% ethanol granule, dry, tabletting, make 150, every tablet weight 0.3g.
After testing, in finished product, the content of glycyrrhizic acid is 56.2mg/ sheet.
Embodiment 3
Take 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 CO2In supercritical extraction device, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 220min, obtain supercritical extract, standby;Take all the other Chinese medicines, pulverize, add 50% ethanol of 5L, put into and microwave extracting apparatus carries out microwave extracting, extract power 800W, extract 2 times, each 10 minutes, combining extraction liquid, concentration, it is added on Dl01 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain microwave extraction thing, standby;Above-mentioned supercritical extract and microwave extraction thing are mixed, adds starch, 70% ethanol granule, dry, tabletting, make 150, every tablet weight 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 Mouse Gastric Cancer cell MFC cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse Gastric Cancer cell MFC cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Armeniaca mume Sieb. common cold tablet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh 100mg Armeniaca mume Sieb. common cold 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 reagents
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137);Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419);NaHC03(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 equipments
Lycra inverted microscope (Germany Leica model: DMIL);Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190);C02 incubator (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 (Gilson Inc of France model: P2);Electronic balance (Sai Duolisi company limited of Germany model: BT323S);Full-automatic high-pressure autoclave (SANYO company of Japan model: MLS-3020);Table electrothermal air dry oven (Shanghai precision 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 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) MFC cell DMEM+10%FBS is in 37 DEG C, 5%CO2Carry out cellar culture (10cm culture dish), when Growth of Cells to logarithmic (log) phase, collect cell, discard culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, it is added thereto in 5ml complete medium and reaction, is proceeded in centrifuge tube after piping and druming cell, 1000rpm is centrifuged 5min, adjusts concentration of cell suspension 3 × 104Individual/ml.
2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, and culture plate puts into (37 DEG C, 5%C02) cellar culture in cell culture incubator.
3) according to cell growth status, general long to 50%-70%, add Armeniaca mume Sieb. common cold 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, made crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) arranging background (being not added with cell, only add culture fluid), control wells (cell, the medicine dissolution medium of same concentrations, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes simultaneously.
7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repeats 3 times.
3. statistical disposition
Adopting 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 MFC 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 Armeniaca mume Sieb. common cold tablet is to MFC cell inhibitory effect influence research (X ± SD)
Group |
Drug level (mg/ml) |
Suppression ratio (%) |
Matched group |
0 |
0 |
1 |
5 |
8.52±7.36 |
2 |
10 |
18.26±13.19* |
3 |
15 |
30.19±16.92** |
4 |
20 |
45.26±18.31** |
Note: compare with matched group, * P is < O.01;* P < 0.001.
5. experiment conclusion
The Armeniaca mume Sieb. common cold tablet of the present invention can suppress MFC cell proliferation, reduces the Growth of Cells number of MFC cell, and this effect is dose dependent.