CN103735961A - Application of boenninghausenia sessilicarpa cough-asthma tablet to prepare medicaments for inhibiting lymphoma cell YAC-1 proliferation - Google Patents
Application of boenninghausenia sessilicarpa cough-asthma tablet to prepare medicaments for inhibiting lymphoma cell YAC-1 proliferation Download PDFInfo
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- CN103735961A CN103735961A CN201310686375.3A CN201310686375A CN103735961A CN 103735961 A CN103735961 A CN 103735961A CN 201310686375 A CN201310686375 A CN 201310686375A CN 103735961 A CN103735961 A CN 103735961A
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- 206010011224 Cough Diseases 0.000 title claims abstract description 47
- 208000006673 asthma Diseases 0.000 title claims abstract description 46
- 239000003814 drug Substances 0.000 title claims abstract description 30
- 101000981253 Mus musculus GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1 Proteins 0.000 title claims abstract description 18
- 206010025323 Lymphomas Diseases 0.000 title claims abstract description 13
- 230000002401 inhibitory effect Effects 0.000 title abstract description 5
- 241000772044 Boenninghausenia Species 0.000 title abstract 7
- 230000035755 proliferation Effects 0.000 title abstract 3
- 238000002360 preparation method Methods 0.000 claims abstract description 25
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- 238000000874 microwave-assisted extraction Methods 0.000 claims abstract description 15
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- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 abstract description 12
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 abstract description 12
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 abstract description 12
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 abstract description 12
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 abstract description 12
- 229940025878 hesperidin Drugs 0.000 abstract description 12
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 abstract description 12
- 229940126680 traditional chinese medicines Drugs 0.000 abstract description 2
- 244000001632 Acorus gramineus Species 0.000 abstract 1
- 235000013073 Acorus gramineus Nutrition 0.000 abstract 1
- 240000000691 Houttuynia cordata Species 0.000 abstract 1
- 235000013719 Houttuynia cordata Nutrition 0.000 abstract 1
- 241001269715 Laggera pterodonta Species 0.000 abstract 1
- 240000001341 Reynoutria japonica Species 0.000 abstract 1
- 235000018167 Reynoutria japonica Nutrition 0.000 abstract 1
- 241000417534 Stemona sessilifolia Species 0.000 abstract 1
- 229960004756 ethanol Drugs 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000000243 solution Substances 0.000 description 10
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010062717 Increased upper airway secretion Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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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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- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 description 1
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of traditional Chinese medicines, and specifically relates to application of a boenninghausenia sessilicarpa cough-asthma tablet to prepare medicaments for inhibiting lymphoma cell YAC-1 proliferation and a preparation method for the boenninghausenia sessilicarpa cough-asthma tablet. The boenninghausenia sessilicarpa cough-asthma tablet provided by the invention possesses the application to prepare medicaments for inhibiting lymphoma cell YAC-1 proliferation. The boenninghausenia sessilicarpa cough-asthma tablet is prepared from the following bulk drugs: 156 g of dried orange peel, 96 g of acorus gramineus, 312 g of reynoutria japonica, 188 g of radix asparagi, 375 g of boenninghausenia sessilicarpa, 96 g of stemona sessilifolia, 750 g of caulis marsdeniae tenacissimae, 375 g of laggera pterodonta, 96 g of semen armeniacae Amarum., 375 g of houttuynia cordata and 188 g of cortex mori. Supercritical extraction and microwave extraction are employed for preparing the boenninghausenia sessilicarpa cough-asthma tablet, and thus hesperidin content is substantially improved.
Description
Technical field
The invention belongs to technical field of Chinese medicines, being specifically related to a kind of Herba Boenninghauseniae cough with asthma sheet, in preparation, to suppress lymphoma cell YAC-1 thin
Born of the same parents breed application in medicine and the preparation method of Herba Boenninghauseniae cough with asthma sheet.
Background technology
Herba Boenninghauseniae cough with asthma granule standard No. WS-10449(ZD-0449)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.By Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, as crude drug, made, there is the effect of removing heat-phlegm, relieving cough and asthma.The cough expectorant that causes for lung-heat is thick, xerostomia itching throat, and the phlegm-damp cough with asthma that causes of acute/chronic bronchitis.
In prior art, the report that not yet has the application of Herba Boenninghauseniae cough with asthma granule in suppress lymphoma cell YAC-1 cell proliferation medicine in preparation, also there are no Herba Boenninghauseniae cough with asthma granule, extract the report that preparation aspect adopts 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 Herba Boenninghauseniae cough with asthma sheet to suppress the application in lymphoma cell YAC-1 cell proliferation medicine in preparation.
Another object of the present invention is to provide a kind of preparation method of Herba Boenninghauseniae cough with asthma sheet.
The object of the invention is to realize by following scheme:
Herba Boenninghauseniae cough with asthma sheet suppresses the application in lymphoma cell YAC-1 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made as crude drug by Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, the preparation method of described Herba Boenninghauseniae cough with asthma sheet is comprised of the following step: get Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae, 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 180-220min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
Preferably, above-mentioned Herba Boenninghauseniae cough with asthma sheet suppresses the application in lymphoma cell YAC-1 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made as crude drug by Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, the preparation method of described Herba Boenninghauseniae cough with asthma sheet is comprised of the following step: get Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae, 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 200min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
In prior art, the each 8-16g of Herba Boenninghauseniae cough with asthma granule, 3-4 time on the one.Herba Boenninghauseniae cough with asthma granule dosage is large, and granule preparation need to add a large amount of sugar, and 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.The every heavy 0.5g of Herba Boenninghauseniae cough with asthma sheet that adopts the inventive method to be prepared into only needs 2-4 sheet at every turn, within 1st, takes 3-4 time.Greatly reduced dose having under the condition of more active component.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 of content of hesperidin in Herba Boenninghauseniae cough with asthma sheet prepared by test one, distinct methods
L, instrument and reagent Herba Boenninghauseniae cough with asthma of the present invention sheet: press embodiment 1 method preparation, use 3007g crude drug, make 250 through extracting, every heavy 0.5g.Former Herba Boenninghauseniae cough with asthma granule, according to WS-10449(ZD-0449)-2002 standard method preparations.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Hesperidin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
According to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010), measure.
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filler; Acetonitrile-0.1% phosphoric acid solution (24: 76) is mobile phase, and detection wavelength is 283nm.Number of theoretical plate calculates and should be not less than 4000 by Hesperidin peak.
It is appropriate that Hesperidin reference substance is got in the preparation of reference substance solution, accurately weighed, adds methanol and make every 1ml containing the solution of 60 μ g, obtains.
Herba Boenninghauseniae cough with asthma sheet of the present invention is got in the preparation of need testing solution, and porphyrize, gets 0.25g, accurately weighed, puts in conical flask, precision adds methanol 25ml, weighed weight, and reflux 30 minutes, lets cool, more weighed weight, with methanol, supply less loss weight, filter, get subsequent filtrate, obtain.
The Herba Boenninghauseniae cough with asthma granule of this contrast is got in the preparation of reference product need testing solution, and porphyrize, gets 4g, accurately weighed, puts in conical flask, precision adds methanol 25ml, weighed weight, and reflux 30 minutes, lets cool, more weighed weight, with methanol, supply less loss weight, filter, get subsequent filtrate, obtain.
The content under this product content uniformity item is got in the preparation of need testing solution, and porphyrize, mixes, and algoscopy is accurate reference substance solution and each 5 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains
3, result
Result shows, in Herba Boenninghauseniae cough with asthma sheet of the present invention, the content of Hesperidin is 15-30mg/ sheet; And the content of Hesperidin is 3.2mg/ bag in former Herba Boenninghauseniae cough with asthma granule, each serving the content of hesperidin of 2 of consumptions be former granule content 5-10 doubly, in the situation that dose reduces, content of hesperidin improves a lot.
Above-mentioned research shows, the Herba Boenninghauseniae cough with asthma sheet that adopts preparation method of the present invention to prepare, active constituent content is far away higher than WS-10449(ZD-0449) the Herba Boenninghauseniae cough with asthma granule prepared of the method recorded of-2002 standards.
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 Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae 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 200min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
After testing, in finished product, the content of Hesperidin is 59.6mg/ sheet.
Embodiment 2
Get Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae 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 180min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
After testing, in finished product, the content of Hesperidin is 32.9mg/ sheet.
Embodiment 3
Get Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae 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 220min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
After testing, in finished product, the content of Hesperidin is 44.7mg/ sheet.
Embodiment 4: Herba Boenninghauseniae cough with asthma sheet suppresses the experimentation data of mouse lymphoma cell YAC-1 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse lymphoma cell YAC-1 cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Herba Boenninghauseniae cough with asthma sheet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg Herba Boenninghauseniae cough with asthma sheet, 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 dehydrated alcohol in order to the use of matched group simultaneously.
1.3 experiment reagent
DMEM (Cat.No.12100-061Lot.No.758137 of GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); NaHC0
3(Cat.No.11810-033Lot.No.1088387 of Shanghai Jiu Yi chemical reagent company limited); 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): the autogamy of PBS(laboratory);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX190); C0
2incubator (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. Sprlng company model: S/N020579); 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) YAC-1 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%C02, when Growth of Cells is during to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml0.25% 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 Herba Boenninghauseniae cough with asthma sheet 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 low-speed oscillation 10min on shaking table, as entered 200 μ l dimethyl sulfoxide, is put in every hole, 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 disposition
Adopt correlation analysis and Student t check in Microsoft Excel2007 software, data represent with mean ± SD.
4. experimental result
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 Herba Boenninghauseniae cough with asthma sheet is on YAC-1 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 10.31±7.66 |
| 2 | 10 | 18.69±9.71* |
| 3 | 15 | 28.34±12.96** |
| 4 | 20 | 39.64±14.03** |
Note: with matched group comparison, * P<O.01; * P<0.001.
5. experiment conclusion
Herba Boenninghauseniae cough with asthma sheet of the present invention can suppress YAC-1 cell proliferation, reduces the Growth of Cells number of YAC-1 cell, and this effect is dose dependent.
Claims (2)
1. Herba Boenninghauseniae cough with asthma sheet suppresses the application in lymphoma cell YAC-1 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made as crude drug by Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, it is characterized in that, the preparation method of described Herba Boenninghauseniae cough with asthma sheet is comprised of the following step: get Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae, 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 180-220min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
2. according to the Herba Boenninghauseniae cough with asthma sheet described in claim l, in preparation, suppress the application in lymphoma cell YAC-1 cell proliferation medicine, described Herba Boenninghauseniae cough with asthma sheet is made as crude drug by Pericarpium Citri Reticulatae 156g, Rhizoma Acori Graminei 96g, Rhizoma Polygoni Cuspidati 312g, Radix Asparagi 188g, Herba Boenninghauseniae 375g, Radix Stemonae 96g, Caulis Marsdeniae Tenacissimae 750g, Herba laggera 375g, Semen Armeniacae Amarum 96g, Herba Houttuyniae 375g, Cortex Mori 188g, it is characterized in that, the preparation method of described Herba Boenninghauseniae cough with asthma sheet is comprised of the following step: get Rhizoma Acori Graminei, Herba laggera, Herba Boenninghauseniae, Herba Houttuyniae, 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 200min, obtains supercritical extract, standby; Get all the other Chinese medicines, pulverize, add 50% ethanol of 5L, 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, standby; Above-mentioned supercritical extract and microwave extraction thing are mixed, add starch, 70% ethanol granule processed, dry, tabletting, makes 250, every heavy 0.5g.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628770A (en) * | 2004-08-27 | 2005-06-22 | 唐秋海 | Cough stopping and asthma relieving medicine and its preparation |
| CN101015645A (en) * | 2007-03-09 | 2007-08-15 | 阮玉江 | Medicine for treating cancer and preparing method therefor |
| CN102133363A (en) * | 2011-03-22 | 2011-07-27 | 唐秋海 | Quality detection method for chinarue cough particle |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628770A (en) * | 2004-08-27 | 2005-06-22 | 唐秋海 | Cough stopping and asthma relieving medicine and its preparation |
| CN101015645A (en) * | 2007-03-09 | 2007-08-15 | 阮玉江 | Medicine for treating cancer and preparing method therefor |
| CN102133363A (en) * | 2011-03-22 | 2011-07-27 | 唐秋海 | Quality detection method for chinarue cough particle |
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| Title |
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| 贾玉梅等: "基于二苯乙烯类为主要活性成分的虎杖药理作用研究进展", 《中国实验方剂学杂志》 * |
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