CN102988748A - 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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- CN102988748A CN102988748A CN2012103769539A CN201210376953A CN102988748A CN 102988748 A CN102988748 A CN 102988748A CN 2012103769539 A CN2012103769539 A CN 2012103769539A CN 201210376953 A CN201210376953 A CN 201210376953A CN 102988748 A CN102988748 A CN 102988748A
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- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 claims description 33
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- 230000005764 inhibitory process Effects 0.000 claims description 3
- UILPJVPSNHJFIK-UHFFFAOYSA-N Paeonol Chemical compound COC1=CC=C(C(C)=O)C(O)=C1 UILPJVPSNHJFIK-UHFFFAOYSA-N 0.000 abstract description 22
- YLTGFGDODHXMFB-UHFFFAOYSA-N isoacetovanillon Natural products COC1=CC=C(C(C)=O)C=C1O YLTGFGDODHXMFB-UHFFFAOYSA-N 0.000 abstract description 11
- MLIBGOFSXXWRIY-UHFFFAOYSA-N paeonol Natural products COC1=CC=C(O)C(C(C)=O)=C1 MLIBGOFSXXWRIY-UHFFFAOYSA-N 0.000 abstract description 11
- 241000045403 Astragalus propinquus Species 0.000 abstract 1
- 241000007126 Codonopsis pilosula Species 0.000 abstract 1
- 241000112528 Ligusticum striatum Species 0.000 abstract 1
- 241001648835 Polygonum cuspidatum Species 0.000 abstract 1
- 235000018167 Reynoutria japonica Nutrition 0.000 abstract 1
- 240000006079 Schisandra chinensis Species 0.000 abstract 1
- 235000008422 Schisandra chinensis Nutrition 0.000 abstract 1
- 235000006533 astragalus Nutrition 0.000 abstract 1
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- 239000013558 reference substance Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
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- JWHHANVGNNWIRI-UHFFFAOYSA-N methanol phosphoric acid hydrate Chemical compound O.OC.OP(O)(O)=O JWHHANVGNNWIRI-UHFFFAOYSA-N 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
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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 the Chinese medicine preparation technical field, be specifically related to a kind of preparation method and application of yin nourishing metformin.
Background technology
The yin nourishing metformin is recorded in Ministry of Public Health standard WS3-B-0984-91, 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, the energy Replenishing QI and nourishing YIN, heat clearing and blood circulation promoting.Be used for diabetes.
In the prior art, not yet there is the yin nourishing metformin 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 yin nourishing metformin.
Another object of the present invention is to provide the application of a kind of yin nourishing metformin in preparation inhibition human promyelocytic leukemia cell HL-60 cell proliferation medicine.
Technical scheme: the objective 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 the 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 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 yin nourishing metformin, 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 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.
The application of above-mentioned yin nourishing metformin in preparation inhibition human promyelocytic leukemia cell HL-60 cell proliferation medicine.
In the 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, takes 3 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 paeonol content in the yin nourishing metformin of test one, distinct methods preparation
1, instrument and reagent yin nourishing metformin of the present invention: press the preparation of embodiment 3 methods, use the 1665g crude drug, make 1000 through extraction, every heavy 0.5g.Former yin nourishing metformin by the preparation of ministry standard method, uses the 1665g crude drug, makes 1000 through extraction, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; The METTLERAE240 electronic analytical balance; Paeonol 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 paeonol peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes by weighing at 4 hours paeonol 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 yin nourishing metformin of the present invention and former yin nourishing metformin, 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 that the content of paeonol is the 1.24mg/ sheet in the yin nourishing metformin of the present invention; And the content of paeonol is the 0.18mg/ sheet in the former yin nourishing metformin, and in the situation that dose reduces, paeonol content improves a lot.
Above-mentioned studies show that, the yin nourishing metformin that adopts the present invention to prepare, 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 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 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, with Rhizoma Chuanxiong, join in the 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, get supercritical extract, 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 paeonol is the 1.29mg/ sheet in the finished product.
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, with Rhizoma Chuanxiong, 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 paeonol is the 1.37mg/ sheet in the finished product.
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, with Rhizoma Chuanxiong, 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 paeonol is the 1.24mg/ sheet in the finished product.
Embodiment 4: the yin nourishing metformin suppresses the experimentation data of HL-60 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing 100mg yin nourishing metformin, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ ldoff 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 HL-60 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 yin nourishing metformin 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 HL-60 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 yin nourishing metformin affects the HL-60 cell inhibitory effect and grinds
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
The yin nourishing metformin can suppress the HL-60 cell proliferation, reduces the Growth of Cells number of HL-60 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of a 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, it is characterized in that described method is comprised of the following step: get Rhizoma Chuanxiong, join CO
2In the 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 yin nourishing metformin 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 yin nourishing metformin 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 yin nourishing metformin 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 yin nourishing metformin in preparation inhibition human promyelocytic leukemia cell HL-60 cell proliferation medicine.
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