CN103028005B - Preparation method of anti-lumbago tablet and application thereof - Google Patents
Preparation method of anti-lumbago tablet and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of an anti-lumbago tablet. The anti-lumbago tablet is prepared by using 108g of folium cortex eucommiae fried with salt, 81g of fructus psoraleae fried with salt, 81g of teasel root, 108g of angelica sinensis, 81g of fried rhizoma atractylodis macrocephalae, 81g of the root of bidentate achyranthes, 27g of cinnamon, 27g of prepared frankincense, 81g of prepared rhizoma cibotii, 43g of root of common peony, 54g of rhizoma alismatis and 43g of ground beeltle fried with wine as raw materials; and the anti-lumbago tablet is prepared by using supercritical extraction and microwave-assisted extraction, so that the content of ferulic acid is greatly enhanced. The invention also provides application of the anti-lumbago tablet in preparation of medicine for inhibiting cell proliferation of human kidney cancer cell KETR-3.
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 anti-lumbago tablet.
Background technology
Anti-lumbago tablet is recorded in Ministry of Public Health standard WS3-B-1053-91, made as crude drug by Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g, energy strengthening vital energy of kidney, promoting blood circulation and stopping pain.For lumbago due to renal deficiency, lumbar muscle strain.
In prior art, not yet there is anti-lumbago tablet extracting the report that adopts supercritical and microwave technology 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 anti-lumbago tablet.
Another object of the present invention is to provide a kind of anti-lumbago tablet to suppress the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of anti-lumbago tablet, by Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g makes as crude drug, described method is made up of the following step: get Radix Angelicae Sinensis, Cortex Cinnamomi, Olibanum (processed), 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 DEG C, CO2 flow 1-3m1/g crude drug min, extraction time 150-180min, obtain supercritical extract, for subsequent use, 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, for subsequent use, 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 anti-lumbago tablet, 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 anti-lumbago tablet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of anti-lumbago tablet, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 DEG C of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned anti-lumbago tablet suppresses the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation.
In prior art, every 0.5g of anti-lumbago tablet, each 6,3 times on the one, the every 0.5g of anti-lumbago tablet that adopts the present invention to be prepared into only needs 3 at every turn, within 1st, takes 3 times, under the condition with more active component, has greatly reduced dose.This conclusion can be by following evidence.
The comparison of ferulaic acid content in anti-lumbago tablet prepared by test one, distinct methods
1, instrument and reagent anti-lumbago tablet of the present invention: press embodiment 3 method preparations, use 815g crude drug, make 1000 through extracting, every heavy 0.5g.Former anti-lumbago tablet, by the preparation of ministry standard method, uses 815g crude drug, makes 1000 through extracting, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLER AE240 electronic analytical balance; Ferulic acid 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 ferulic acid peak and is calculated, and should be not less than 3000.
The preparation of reference substance solution: precision takes at 60 DEG C of drying under reduced pressure ferulic acid reference substance of 4 hours appropriate, adds methanol and makes the solution of every 1ml containing 18 μ g, to obtain final product.
The preparation of need testing solution: get anti-lumbago tablet of the present invention and former anti-lumbago tablet, porphyrize, mixes, and gets 1g, accurately weighed, precision adds 70% ethanol 20ml, close plug, supersound process 10 minutes, centrifugal, get supernatant, 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 anti-lumbago tablet of the present invention, the content of ferulic acid is 1.12mg/ sheet; And the content of ferulic acid is 0.14mg/ sheet in former anti-lumbago tablet, in the situation that dose reduces, ferulaic acid content improves a lot.
Above-mentioned research shows, the anti-lumbago tablet that adopts the present invention to prepare, and active constituent content is higher than the standby anti-lumbago tablet of ministry standard legal system.
Detailed description of the invention
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 Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g, by Radix Angelicae Sinensis, Cortex Cinnamomi, Olibanum (processed), join in CO2 supercritical extraction device, ethanol is as entrainer, the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, 30 DEG C of temperature, CO2 flow 1m1/g crude drug min, extraction time 150min, obtain supercritical extract, for subsequent use; 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, for subsequent use; 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 ferulic acid is 1.15mg/ sheet.
Embodiment 2
Get Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g, by Radix Angelicae Sinensis, Cortex Cinnamomi, Olibanum (processed), 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, for subsequent use; 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, for subsequent use; 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 ferulic acid is 1.09mg/ sheet.
Embodiment 3
Get Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g, by Radix Angelicae Sinensis, Cortex Cinnamomi, Olibanum (processed), 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 DEG C of temperature, CO
2flow 2m1/g crude drug min, extraction time 160min, obtains supercritical extract, for subsequent use; 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, for subsequent use; 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 ferulic acid is 1.12mg/ sheet.
Embodiment 4: anti-lumbago tablet suppresses the experimentation data of KETR-3 cell proliferation
1 experiment material
1.1 experiment cell strains
Human renal carcinoma cell KETR-3 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: anti-lumbago tablet of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg anti-lumbago tablet, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ l doff pipe subpackages ,-20 DEG C of storages, 0.2 μ m filter filters the use of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061 Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The NaHCO3(Shanghai hundred million Cat.No.11810-033 Lot.No.1088387 of chemical reagent company limited 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); PBS(laboratory autogamy);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX 190); CO2 incubator (FORMA model: 3111); Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD); Pure water instrument (Spring company of U.S. 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) KETR-3 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 DEG C, 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 DEG C 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, and culture plate is put into cell culture incubator (37 DEG C, 5%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add anti-lumbago 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 every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on 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), 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 inspection 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, in the time that dosage reaches 5mg/ml, to KETR-3 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 anti-lumbago tablet is on KETR-3 cell inhibitory effect impact research
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
Anti-lumbago tablet can suppress KETR-3 cell proliferation, reduces the Growth of Cells number of KETR-3 cell, and this effect is dose dependent.
Claims (4)
1. an anti-lumbago tablet suppresses the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation, it is characterized in that anti-lumbago tablet is made up as crude drug of Cortex Eucommiae (parched with salt) leaf 108g, salt Fructus Psoraleae (parched) 81g, Radix Dipsaci 81g, Radix Angelicae Sinensis 108g, Rhizoma Atractylodis Macrocephalae (parched) 81g, Radix Achyranthis Bidentatae 81g, Cortex Cinnamomi 27g, Olibanum (processed) 27g, Rhizoma Cibotii 81g processed, Radix Paeoniae Rubra 43g, Rhizoma Alismatis 54g, wine Eupolyphaga (parched) 43g, preparation method is made up of the following step: get Radix Angelicae Sinensis, Cortex Cinnamomi, Olibanum (processed), 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 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, for subsequent use; 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, for subsequent use; 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. a kind of anti-lumbago tablet suppresses the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. a kind of anti-lumbago tablet suppresses the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation according to claim 1, it is characterized in that the power of microwave extracting described in preparation method 500W, extracts 6 minutes at every turn.
4. a kind of anti-lumbago tablet suppresses the application in human renal carcinoma cell KETR-3 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the extracting pressure 20MPa of supercritical extraction, 40 DEG C of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
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| CN103638322B (en) * | 2013-11-28 | 2015-12-02 | 上海欧睿生物科技有限公司 | A kind of preparation method of KidneyHarmonizing Pill and application |
| CN103638489B (en) * | 2013-12-04 | 2015-07-15 | 新乡医学院第一附属医院 | Preparation method and application of kidney-tonifying pills |
| CN103705624B (en) * | 2013-12-22 | 2015-09-02 | 河北工程大学 | A kind of preparation method of long-life sheet and application |
| CN104173629A (en) * | 2014-08-16 | 2014-12-03 | 黑龙江江恒医药科技有限公司 | Anti-lumbago tablet and preparation method thereof |
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| CN1748753A (en) * | 2005-10-20 | 2006-03-22 | 北京阜康仁生物制药科技有限公司 | Waist pain preparation and new preparing method |
| CN102085280A (en) * | 2009-12-06 | 2011-06-08 | 湖北广仁药业有限公司 | One-step granulating method of tablets for treating lumbago |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1748753A (en) * | 2005-10-20 | 2006-03-22 | 北京阜康仁生物制药科技有限公司 | Waist pain preparation and new preparing method |
| CN102085280A (en) * | 2009-12-06 | 2011-06-08 | 湖北广仁药业有限公司 | One-step granulating method of tablets for treating lumbago |
Non-Patent Citations (2)
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| 微波辅助萃取技术在中药现代化中的应用;李攻科等;《精细化工》;20071231;第24卷(第12期);第1184页摘要 * |
| 李攻科等.微波辅助萃取技术在中药现代化中的应用.《精细化工》.2007,第24卷(第12期),第1184页摘要. |
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