CN103768211B - A kind of preparation method of Armeniaca mume Sieb. common cold tablet and the application in suppression lymphoma cell EL4 cell proliferation thereof - Google Patents
A kind of preparation method of Armeniaca mume Sieb. common cold tablet and the application in suppression lymphoma cell EL4 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 Armeniaca mume Sieb. common cold tablet.The preparation method of Armeniaca mume Sieb. common cold tablet of the present invention, be 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, adopt supercritical extraction and microwave extracting to be prepared from, glycyrrhizic acid content is improved a lot.Present invention also offers the application of Armeniaca mume Sieb. common cold tablet in preparation suppression mouse lymphoma cell EL4 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 suppressing the application in mouse lymphoma cell EL4 cell proliferation.
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.Be 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, Armeniaca mume Sieb. cold granules is not yet had to extract 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 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 mouse lymphoma cell EL4 cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of Armeniaca mume Sieb. common cold tablet, be 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: 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 DEG C, 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, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 150, the heavy 0.3g of every sheet.
In the preparation method of above-mentioned Armeniaca mume Sieb. common cold tablet, described CO
2in 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 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 DEG C.
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, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned Armeniaca mume Sieb. common cold tablet suppresses the application in mouse lymphoma cell EL4 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 large, and granule preparation needs to add a large amount of sugar, and diabetics is not suitable for, and the words taste of not sugaring is not good, and under patient is not easy clothes, compliance is poor.The heavy 0.3g of the every sheet of the Armeniaca mume Sieb. common cold tablet adopting the inventive method to be prepared into, only needs 2 at every turn, within 1st, takes 2-3 time.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.And being prepared into tablet, patient swallows with water, and 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: by the preparation of embodiment 1 method, use 1510g crude drug, makes 150 through extracting, the heavy 0.3g of every sheet.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 version in 2010 annex VI D).
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; Determined wavelength is 250nm.Number of theoretical plate calculates should be not less than 3000 by glycyrrhizic acid peak.
The preparation precision of reference substance solution takes monoammonium glycyrrhizinate reference substance in right amount, adds 90% methanol and makes 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, put in tool plug conical flask, precision adds 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 gets subsequent filtrate, to obtain final product.
The Armeniaca mume Sieb. cold granules of this contrast, porphyrize are got in the preparation of reference product need testing solution, mixing, 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, supernatant microporous filter membrane (0.45 μm) filters, get subsequent filtrate, to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 20 μ l of need testing solution, injection liquid chromatography, measures, 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, the glycyrrhizic acid content each serving consumption 2 is 5-10 times of former granule content, and when dose reduces, glycyrrhizic acid content improves a lot.
Above-mentioned research shows, adopts Armeniaca mume Sieb. common cold tablet prepared by preparation method of the present invention, 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.
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 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 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, temperature 40 DEG C, 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, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 150, the heavy 0.3g of every sheet.
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 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, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 150, the heavy 0.3g of every sheet.
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 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, temperature 30 DEG C, 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, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 150, the heavy 0.3g of every sheet.
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 lymphoma cell EL4 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse lymphoma cell EL4 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: take 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 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); 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 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) EL4 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 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, 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 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 EL4 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 EL4 cell inhibitory effect influence research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 9.79±8.36 |
| 2 | 10 | 19.38±12.96* |
| 3 | 15 | 38.92±17.09** |
| 4 | 20 | 49.26±19.64** |
Note: compare with matched group, * P<0.01; * P<0.001.
5. experiment conclusion
Armeniaca mume Sieb. common cold tablet of the present invention can suppress EL4 cell proliferation, and reduce the Growth of Cells number of EL4 cell, this effect is dose dependent.
Claims (7)
1. Armeniaca mume Sieb. common cold tablet suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, it is characterized in that, described Armeniaca mume Sieb. common cold tablet 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, the preparation method of described Armeniaca mume Sieb. common cold tablet 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 DEG C, 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, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 150, the heavy 0.3g of every sheet.
2. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
3. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extracting pressure is 25MPa.
4. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, it is characterized in that, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
5. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, it is characterized in that, described CO
2in supercritical extraction, extraction time is 200min.
6. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, and it is characterized in that, described microwave extracting power is 700W.
7. the Armeniaca mume Sieb. common cold tablet according to claim l suppresses the application in mouse lymphoma cell EL4 cell proliferation in preparation, and it is characterized in that, each extraction time of described microwave extracting is 8 minutes.
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