CN102988748B - Preparation method and application of yin tonifying and blood sugar reducing tablet - Google Patents
Preparation method and application of yin tonifying and blood sugar reducing tablet Download PDFInfo
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Abstract
The invention provides a preparation method of a yin tonifying and blood sugar reducing tablet which is prepared from crude drugs including 250g of astragalus mongholicus, 110g of codonopsis pilosula, 145g of radix puerariae, 110g of fructus lycii, 145g of radix scrophulariae, 110g of radix polygonati officinalis, 180g of radix rehmanniae, 110g of rhizome anemarrhenae, 110g of moutan bark, 145g of ligusticum wallichii, 180g of polygonum cuspidatum and 70g of schisandra chinensis through supercritical extraction and microwave extraction, so that the content of paeonol is greatly improved. The invention also provides the application of the yin tonifying and blood sugar reducing tablet in preparing a medicine for restraining proliferation of human promyelocytic leukemia cell, HL-60 cell.
Description
Technical field
The present invention relates to Chinese medicine preparation technical field, be specifically related to a kind of preparation method and application of yin nourishing metformin.
Background technology
Yin nourishing metformin is recorded in Ministry of Public Health standard WS3-B-0984-91, by Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g, as crude drug, made, energy Replenishing QI and nourishing YIN, heat clearing and blood circulation promoting.For diabetes.
In prior art, not yet there is yin nourishing metformin to adopt the report of supercritical and microwave technology extracting aspect preparation, 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 preparation method of yin nourishing metformin.
Another object of the present invention is to provide a kind of yin nourishing metformin to suppress the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of yin nourishing metformin, by Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g makes as crude drug, described method is comprised of the following step: get Rhizoma Chuanxiong, join in CO2 supercritical extraction device, ethanol is as entrainer, the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO2 flow 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, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 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, standby, above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
The preparation method of above-mentioned a kind of yin nourishing metformin, described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
The preparation method of above-mentioned a kind of yin nourishing metformin, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of yin nourishing metformin, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned yin nourishing metformin suppresses the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation.
In prior art, every 0.5g of yin nourishing metformin, each 8,3 times on the one, the every 0.5g of yin nourishing metformin that adopts the present invention to be prepared into only needs 4 at every turn, within 1st, takes 3 times, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of paeonol content in yin nourishing metformin prepared by test one, distinct methods
1, instrument and reagent yin nourishing metformin of the present invention: press embodiment 3 method preparations, use 1665g crude drug, make 1000 through extracting, every heavy 0.5g.Former yin nourishing metformin, by the preparation of ministry standard method, is used 1665g crude drug, through extracting, makes 1000, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLERAE240 electronic analytical balance; Paeonol 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; Detection wavelength is 280nm.Number of theoretical plate is pressed paeonol 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 paeonol reference substance of 4 hours appropriate, adds methanol and makes every 1ml containing the solution of 18 μ g, obtains.
The preparation of need testing solution: get yin nourishing metformin of the present invention and former yin nourishing metformin, porphyrize, mixes, and gets 1g, accurately weighed, and 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 injection liquid chromatography, measures, and obtains.
3, result
Result shows, in yin nourishing metformin of the present invention, the content of paeonol is 1.24mg/ sheet; And the content of paeonol is 0.18mg/ sheet in former yin nourishing metformin, in the situation that dose reduces, paeonol content improves a lot.
Above-mentioned research shows, the yin nourishing metformin that adopts the present invention to prepare, and active constituent content is higher than the standby yin nourishing metformin 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 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 Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g, by Rhizoma Chuanxiong, 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, CO2 flow 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, extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on D101 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of paeonol is 1.29mg/ sheet.
Embodiment 2
Get Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g, by Rhizoma Chuanxiong, join CO
2in 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, obtains 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, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on D101 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of paeonol is 1.37mg/ sheet.
Embodiment 3
Get Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g, by Rhizoma Chuanxiong, join CO
2in 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, obtains 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, extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on D101 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content of paeonol is 1.24mg/ sheet.
Embodiment 4: yin nourishing metformin suppresses the experimentation data of HL-60 cell proliferation
1 experiment material
1.1 experiment cell strains
Human promyelocytic leukemia cell HL-60 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: yin nourishing metformin of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg yin nourishing metformin, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ ldoff pipe packing ,-20 ℃ of storages, 0.2 μ m filter filters dehydrated alcohol in order to the use of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The NaHCO3(Shanghai Jiu Yi chemical reagent Cat.No.11810-033Lot.No.1088387 of company limited); 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: SPECTRAMAX 190); 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/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 (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) HL-60 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells is during to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% 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 3 * 104/ml of concentration of cell suspension.
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%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add yin nourishing metformin 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, 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.At enzyme-linked immunosorbent assay instrument 490nm place, measure the light absorption value in each hole.
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 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 mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to HL-60 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has utmost point significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 yin nourishing metformin affects and grinds HL-60 cell inhibitory effect
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Yin nourishing metformin can suppress HL-60 cell proliferation, reduces the Growth of Cells number of HL-60 cell, and this effect is dose dependent.
Claims (4)
1. a yin nourishing metformin suppresses the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation, it is characterized in that yin nourishing metformin made as crude drug by Radix Astragali 250g, Radix Codonopsis 110g, Radix Puerariae 145g, Fructus Lycii 110g, Radix Scrophulariae 145g, Rhizoma Polygonati Odorati 110g, Radix Rehmanniae 180g, Rhizoma Anemarrhenae 110g, Cortex Moutan 110g, Rhizoma Chuanxiong 145g, Rhizoma Polygoni Cuspidati 180g, Fructus Schisandrae Chinensis 70g, preparation method is comprised of the following step: get Rhizoma Chuanxiong, 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 1-3m1/g crude drug min, extraction time 150-180min, obtains 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, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
2. yin nourishing metformin suppresses the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation according to claim 1, it is characterized in that described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. yin nourishing metformin suppresses the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation according to claim 1, it is characterized in that described microwave extracting power 500W, extracts 6 minutes at every turn.
4. yin nourishing metformin suppresses the application in human promyelocytic leukemia cell HL-60 cell proliferation medicine in preparation according to claim 1, it 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.
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| CN102397461A (en) * | 2011-11-27 | 2012-04-04 | 苏州派腾生物医药科技有限公司 | Preparation method of tablet for women's health and tranquilness |
| CN102614378A (en) * | 2012-04-26 | 2012-08-01 | 陕西方舟制药有限公司 | Yin nourishing and blood sugar lowering Chinese medicinal composition and preparation method as well as detection method thereof |
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| CN102397461A (en) * | 2011-11-27 | 2012-04-04 | 苏州派腾生物医药科技有限公司 | Preparation method of tablet for women's health and tranquilness |
| CN102614378A (en) * | 2012-04-26 | 2012-08-01 | 陕西方舟制药有限公司 | Yin nourishing and blood sugar lowering Chinese medicinal composition and preparation method as well as detection method thereof |
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