CN103768562A - Application of dizziness-relieving and tranquilizing tablet in preparing medicine for inhibiting cell proliferation of leukemia cell L1210 - Google Patents
Application of dizziness-relieving and tranquilizing tablet in preparing medicine for inhibiting cell proliferation of leukemia cell L1210 Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to application of a dizziness-relieving and tranquilizing tablet in preparing a medicine for inhibiting cell proliferation of a leukemia cell L1210, and a preparation method of the dizziness-relieving and tranquilizing tablet. The dizziness-relieving and tranquilizing tablet is prepared from the following bulk drugs: 400g of pyrola, 160g of epimedium, 160g of astragalus membranaceus, 140g of Chinese angelica, 200g of ligusticum wallichii, 240g of kudzuvine root, 120g of fried bighead atractylodes rhizome, 140g of spina date seed, 120g of prepared pinellia ternate, 120g of rhizoma alismatis, 100g of dried ginger, and 100g of liquorice by adopting supercritical extraction and microwave extraction, so that the content of icariin is greatly improved.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind ofly stop dizzy mind tranquilizing tablet and suppress the application in leukaemia L1210 cell proliferation medicine and the preparation method of stopping dizzy mind tranquilizing tablet in preparation.
Background technology
Stop dizzy mind easing standard No. WS-11411(ZD-1411)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Made as crude drug by Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, there is invigorating the liver and kidney, replenishing QI and blood, effect of tranquilizing mind.For deficiency of the liver and kindey, dizzy due to deficiency of both QI and blood, tinnitus, insomnia, cardiopalmus.
In prior art, not yet have and stop dizzy mind easing at the report that suppresses the application in leukaemia L1210 cell proliferation medicine in preparation, also there are no the report that stops dizzy mind easing extraction preparation aspect employing supercritical and microwave technology, and volatile oil extracts, the method that decocting boils, technique is coarse, backward, 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
Goal of the invention: the object of the present invention is to provide a kind of application of dizzy mind tranquilizing tablet in preparation inhibition leukaemia L1210 cell proliferation medicine that stop.
Another object of the present invention is to provide a kind of preparation method of stopping dizzy mind tranquilizing tablet.
The object of the invention is to realize by following scheme:
Stop dizzy mind tranquilizing tablet and suppress the application in leukaemia L1210 cell proliferation medicine in preparation, describedly stop dizzy mind tranquilizing tablet and made as crude drug by Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, described preparation method of stopping dizzy mind tranquilizing tablet is made up of the following step: get Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae, 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 l-3ml/g crude drug min, extraction time 300-340min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, 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 Diaion HP-10 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 200, every heavy 0.5g.
Preferably, the above-mentioned dizzy mind tranquilizing tablet that stops suppresses the application in leukaemia L1210 cell proliferation medicine in preparation, describedly stop dizzy mind tranquilizing tablet and made as crude drug by Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, described preparation method of stopping dizzy mind tranquilizing tablet is made up of the following step: get Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae, 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 15-30MPa, temperature 30-50 ℃, CO
2flow l-3ml/g crude drug min, extraction time 320min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 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 200, every heavy 0.5g.
In prior art, stop each 1 bag of dizzy mind easing, every bag heavy 5g(Sugarless type) or 10g(have sugared type), 3 times on the one.Stop dizzy mind easing dosage large, and granule preparation need to add a large amount of sugar, diabetics is not suitable for, and the words taste of not sugaring is not good, and patient is not easy under clothes, and compliance is poor.Adopt every of the dizzy mind tranquilizing tablet that stops that the inventive method is prepared into weigh 0.5g, only need 2 at every turn, within 1st, take 3 times.Under the condition with more active component, greatly reduce dose.This conclusion can be by following evidence.And be prepared into tablet, patient swallows with water, dosage is little and without bitterness, patient is easy to accept.
The comparison that stops Icariin content in dizzy mind tranquilizing tablet prepared by test one, distinct methods
L, instrument and reagent the present invention stop dizzy mind tranquilizing tablet: by embodiment 1 method preparation, use 2160g crude drug, make 200 through extracting, every heavy 0.5g.Formerly stop dizzy mind easing, according to WS-11411(ZD-1411)-2002 standard methods preparations.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Icariin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Acetonitrile-water (26: 74) is mobile phase; Detection wavelength is 270nm.Number of theoretical plate calculates and should be not less than 8000 by icariin peak.
The preparation precision of reference substance solution takes at 105 ℃ of icariin reference substances that are dried to constant weight appropriate, adds 70% ethanol and makes the solution of every 1ml containing 50 μ g, to obtain final product.
The dizzy mind tranquilizing tablet that stops of the present invention is got in the preparation of need testing solution, and porphyrize, gets 0.4g, accurately weighed, to put in tool plug conical flask, precision adds 70% ethanol 25ml, weighed weight, close plug, placement is spent the night, supersound process 30 minutes, lets cool, more weighed weight, the weight of supplying less loss with 70% ethanol, shakes up, and with microporous filter membrane, (0.45 μ m) filters, get subsequent filtrate, to obtain final product.
The dizzy mind easing of stopping of this contrast is got in the preparation of reference product need testing solution, and porphyrize, gets 4g, put in tool plug conical flask, precision adds 70% ethanol 25ml, weighed weight, close plug, placement is spent the night, supersound process 30 minutes, let cool, more weighed weight, supply the weight of less loss with 70% ethanol, shake up, with microporous filter membrane, (0.45 μ m) filters, and gets subsequent filtrate, 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, the content that the present invention stops icariin in dizzy mind tranquilizing tablet is 3.5-6g/ sheet; And the former content that stops icariin in dizzy mind easing is 1.2mg/ bag, be 6-10 times of former granule content each serving the Icariin content of 2 of consumptions, in the situation that dose reduces, Icariin content improves a lot.
Above-mentioned research shows, adopts the dizzy mind tranquilizing tablet that stops prepared by preparation method of the present invention, and active constituent content is far away higher than WS-11411(ZD-1411) method recorded of-2002 standards prepare stop dizzy mind easing.
The specific embodiment
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 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 Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae are joined to 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, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 320min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, 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 Diaion HP-10 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 200, every heavy 0.5g.
After testing, in finished product, the content of icariin is 5.9mg/ sheet.
Embodiment 2
Get Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae are joined to 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 300min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, 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 Diaion HP-10 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 200, every heavy 0.5g.
After testing, in finished product, the content of icariin is 3.6mg/ sheet.
Embodiment 3
Get Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae are joined to 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, 30 ℃ of temperature, CO
2flow 1ml/g crude drug min, extraction time 340min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, 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 Diaion HP-10 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 200, every heavy 0.5g.
After testing, in finished product, the content of icariin is 4.5mg/ sheet.
Embodiment 4: stop the experimentation data that dizzy mind tranquilizing tablet suppresses mouse leukemia cell L1210 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse leukemia cell L1210 cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention stops dizzy mind tranquilizing tablet: by embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg and stop dizzy mind tranquilizing tablet, be dissolved in 5ml dehydrated alcohol, 0.2 μ m filter filters, 500 μ ldoff pipe subpackages ,-20 ℃ of storages, 0.2 μ m filter filters the use of dehydrated alcohol in order to matched group simultaneously.
1.3 experiment reagent
DMEM (the Cat.No.12100-061 Lot.No.758137 of GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); NaHC0
3(the Shanghai hundred million Cat.No.11810-033 Lot.No.1088387 of chemical reagent company limited of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); Penicillin G Sodium Salt(AMRESCO company 1); Streptomycin Sulfate (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: SPECTRAMAX 190); C0
2incubator (FORMA model: 3111); Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD); Pure water instrument (Sprlng 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); 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) L1210 cell uses DMEM+10%FBS in 37 ℃, 5%C0
2carry out cellar culture (10cm culture dish), 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 concentration of cell suspension 3 × 10
4individual/ml.
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%C0
2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add and stop dizzy mind tranquilizing 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 low-speed oscillation 10min on shaking table, as entered 200 μ l dimethyl sulfoxide, is put in every hole, 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 disposition
Adopt correlation analysis and Student t check in Microsoft Excel 2007 softwares, data represent with mean ± SD.
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, in the time that dosage reaches 5mg/ml, to L1210 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 stops dizzy mind tranquilizing tablet to L1210 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 12.29±5.72 |
| 2 | 10 | 21.46±8.53* |
| 3 | 15 | 35.39±12.64** |
| 4 | 20 | 45.87±16.98** |
Note: with matched group comparison, * P<O.01; * P<0.001.
5. experiment conclusion
Of the present inventionly stop dizzy mind tranquilizing tablet and can suppress L1210 cell proliferation, reduce the Growth of Cells number of L1210 cell, this effect is dose dependent.
Claims (2)
1. stop dizzy mind tranquilizing tablet and suppress the application in leukaemia L1210 cell proliferation medicine in preparation, describedly stop dizzy mind tranquilizing tablet and made as crude drug by Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, it is characterized in that, described preparation method of stopping dizzy mind tranquilizing tablet is made up of the following step: get Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae, 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 l-3ml/g crude drug min, extraction time 300-340min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, 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 Diaion HP-10 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 200, every heavy 0.5g.
2. suppress the application in leukaemia L1210 cell proliferation medicine according to the dizzy mind tranquilizing tablet that stops described in claim l in preparation, describedly stop dizzy mind tranquilizing tablet and made as crude drug by Herba Pyrolae 400g, Herba Epimedii 160g, Radix Astragali 160g, Radix Angelicae Sinensis 140g, Rhizoma Chuanxiong 200g, Radix Puerariae 240g, Rhizoma Atractylodis Macrocephalae (parched) 120g, Semen Ziziphi Spinosae 140g, Rhizoma Pinelliae Preparata 120g, Rhizoma Alismatis 120g, Rhizoma Zingiberis 100g, Radix Glycyrrhizae 100g, it is characterized in that, described preparation method of stopping dizzy mind tranquilizing tablet is made up of the following step: get Rhizoma Zingiberis, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae (parched), Radix Angelicae Sinensis, Herba Pyrolae, 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 15-30MPa, temperature 30-50 ℃, CO
2flow l-3ml/g crude drug min, extraction time 320min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 6.5L50% ethanol, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extracts 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 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 200, every heavy 0.5g.
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| 国家药品监督管理局: "止眩安神颗粒", 《国家药品标准（试行）颁布件》 * |
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