CN102988881B - Preparation method and application of pulse unblocking tablet - Google Patents
Preparation method and application of pulse unblocking tablet Download PDFInfo
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- CN102988881B CN102988881B CN201210389720.2A CN201210389720A CN102988881B CN 102988881 B CN102988881 B CN 102988881B CN 201210389720 A CN201210389720 A CN 201210389720A CN 102988881 B CN102988881 B CN 102988881B
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Abstract
The invention provides a preparation method of a pulse unblocking tablet. The pulse unblocking tablet is prepared by using 250g of danshen roots, 62.5g of safflower, 125g of radix curcumae, 125g of rehmannia, 62.5g of lignum dalbergiae odoriferae, 62.5g of ligusticum wallichii, 31.5g of frankincense and 31.56g of myrrh as bulk drugs and adopting supercritical extraction and microwave-assisted extraction modes. By adpoting the method, the content of tanshinone is greatly improved; and the invention also provides application of the pulse unblocking tablet in preparation of drugs for inhibiting cell proliferation of mice lymphoma cell YAC-1.
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 tongmailing tablet.
Background technology
Tongmailing tablet is recorded in Ministry of Public Health standard WS3-B-0615-91, by Radix Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, as crude drug, made, energy blood circulation promoting and blood stasis dispelling, coronary circulation-promoting pain-relieving.For angina cordis and myocardial infarction.
In prior art, not yet there is tongmailing tablet 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 tongmailing tablet.
Another object of the present invention is to provide a kind of tongmailing tablet to suppress the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
A kind of preparation method of tongmailing tablet, by Radix Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, as crude drug, made, described method is comprised of the following step: get Flos Carthami, Radix Curcumae, Lignum Dalbergiae Odoriferae, Rhizoma Chuanxiong, Olibanum (processed), Myrrha (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 ℃, 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.
The preparation method of above-mentioned a kind of tongmailing 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 tongmailing tablet, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of above-mentioned a kind of tongmailing tablet, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned tongmailing tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation.
In prior art, every 0.5g of tongmailing tablet, each 5,3 times on the one, the every 0.5g of tongmailing tablet that adopts the present invention to be prepared into only needs 3 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 TANSHINONES content in tongmailing tablet prepared by test one, distinct methods
1, instrument and reagent tongmailing tablet of the present invention: press embodiment 3 method preparations, use 750g crude drug, make 1000 through extracting, every heavy 0.5g.Former tongmailing tablet, by the preparation of ministry standard method, is used 750g crude drug, through extracting, makes 1000, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLERAE240 electronic analytical balance; TANSHINONES 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 calculates by TANSHINONES peak, should be not less than 3000.
The preparation of reference substance solution: precision takes at 60 ℃ of drying under reduced pressure TANSHINONES 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 tongmailing tablet of the present invention and former tongmailing tablet, porphyrize, mixes, and gets 1g, accurately weighed, 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 tongmailing tablet of the present invention, the content of TANSHINONES is 1.47mg/ sheet; And the content of TANSHINONES is 0.32mg/ sheet in former tongmailing tablet, in the situation that dose reduces, TANSHINONES content improves a lot.
Above-mentioned research shows, the tongmailing tablet that adopts the present invention to prepare, and active constituent content is higher than the standby tongmailing tablet 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 Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, by Flos Carthami, Radix Curcumae, Lignum Dalbergiae Odoriferae, Rhizoma Chuanxiong, Olibanum (processed), Myrrha (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 ℃ of temperature, CO
2flow 1m1/g crude drug min, extraction time 150min, 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 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 TANSHINONES is 1.51mg/ sheet.
Embodiment 2
Get Radix Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, by Flos Carthami, Radix Curcumae, Lignum Dalbergiae Odoriferae, Rhizoma Chuanxiong, Olibanum (processed), Myrrha (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, 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 TANSHINONES is 1.54mg/ sheet.
Embodiment 3
Get Radix Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, by Flos Carthami, Radix Curcumae, Lignum Dalbergiae Odoriferae, Rhizoma Chuanxiong, Olibanum (processed), Myrrha (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 ℃ 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 TANSHINONES is 1.47mg/ sheet.
Embodiment 4: tongmailing tablet suppresses the experimentation data of YAC-1 cell proliferation
1 experiment material
1.1 experiment cell strains
Mouse lymphoma cell YAC-1 cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: tongmailing tablet of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take 100mg tongmailing tablet, 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) YAC-1 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 tongmailing 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, 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 YAC-1 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 tongmailing tablet is on YAC-1 cell inhibitory effect impact research
Note: with matched group comparison, * P < 0.01; * P < 0.001
5 experiment conclusion
Tongmailing tablet can suppress YAC-1 cell proliferation, reduces the Growth of Cells number of YAC-1 cell, and this effect is dose dependent.
Claims (4)
1. tongmailing tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation, it is characterized in that tongmailing tablet made as crude drug by Radix Salviae Miltiorrhizae 250g, Flos Carthami 62.5g, Radix Curcumae 125g, Radix Rehmanniae 125g, Lignum Dalbergiae Odoriferae 62.5g, Rhizoma Chuanxiong 62.5g, Olibanum (processed) 31.5g, Myrrha (processed) 31.56g, preparation method is comprised of the following step: get Flos Carthami, Radix Curcumae, Lignum Dalbergiae Odoriferae, Rhizoma Chuanxiong, Olibanum (processed), Myrrha (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 ℃, 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. tongmailing tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in the preparation method of tongmailing tablet
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. tongmailing tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation according to claim 1, it is characterized in that microwave extracting power 500W in the preparation method of tongmailing tablet, extracts 6 minutes at every turn.
4. tongmailing tablet suppresses the application in mouse lymphoma cell YAC-1 cell proliferation medicine in preparation according to claim 1, it is characterized in that CO in the preparation method of tongmailing tablet
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
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