CN103800770A - Application of boenninghausenia cough tablet in preparing medicine for inhibiting cell proliferation of breast tumor cell C127 - Google Patents
Application of boenninghausenia cough tablet in preparing medicine for inhibiting cell proliferation of breast tumor cell C127 Download PDFInfo
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- CN103800770A CN103800770A CN201310669719.XA CN201310669719A CN103800770A CN 103800770 A CN103800770 A CN 103800770A CN 201310669719 A CN201310669719 A CN 201310669719A CN 103800770 A CN103800770 A CN 103800770A
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to application of a boenninghausenia cough tablet in preparing a medicine for inhibiting cell proliferation of a breast tumor cell C127 and a preparation method of the boenninghausenia cough tablet. According to the application of the boenninghausenia cough tablet in preparing the medicine for inhibiting cell proliferation of a breast tumor cell C127, the boenninghausenia cough tablet is prepared from the following bulk drugs: 156g of dried tangerine peel, 96g of rhizoma acori graminei, 312g of polygonum cuspidatum, 188g of asparagus fern, 375g of boenninghausenia, 96g of stemona tuber, 750g of marsdenia tenacissima, 375g of laggera pterodonta, 96g of bitter apricot kernel, 375g of houttuynia cordata, and 188g of white mulberry root-bark. The boenninghausenia cough tablet is prepared by adopting supercritical extraction and microwave extraction, so that the content of hesperidin can be greatly increased.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Herba Boenninghauseniae cough with asthma sheet and suppress application in breast tumor cell C127 cell proliferation medicine and the preparation method of Herba Boenninghauseniae cough with asthma sheet in preparation.
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.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, there is 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, not yet there is Herba Boenninghauseniae cough with asthma granule at the report that suppresses the application in breast tumor cell C127 cell proliferation medicine in preparation, also extract the report of preparation aspect employing supercritical and microwave technology there are no Herba Boenninghauseniae cough with asthma granule, 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 breast tumor cell C127 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 breast tumor cell C127 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made up as crude drug of 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 made up 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, for subsequent use; 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, for subsequent use; 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 breast tumor cell C127 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made up as crude drug of 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 made up 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, for subsequent use; 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, for subsequent use; 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.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 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
Measure according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010).
Chromatographic condition and system suitability are 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 the solution of every 1ml containing 60 μ g, to obtain final product.
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, supply less loss weight with methanol, filter, get subsequent filtrate, to obtain final product.
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, supply less loss weight with methanol, filter, get subsequent filtrate, to obtain final product.
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 the each 5 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and to obtain final product
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, be 5-10 times of former granule content each serving the content of hesperidin of 2 of consumptions, 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, for subsequent use; 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, for subsequent use; 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, for subsequent use; 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, for subsequent use; 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, for subsequent use; 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, for subsequent use; 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 mammary tumor cell C127 cell proliferation
1. experiment material
1.1 experiment cell strains
Mouse mammary tumor cell C127 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 the use of dehydrated alcohol in order to 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); NaHC03 (Shanghai hundred million Cat.No.11810-033Lot.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: SPECTRAMAX190); 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/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) C127 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 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, 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 Excel2007 software, 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 C127 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 Herba Boenninghauseniae cough with asthma sheet is on C127 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 8.25±4.97 |
| 2 | 10 | 15.96±7.12* |
| 3 | 15 | 26.87±11.03** |
| 4 | 20 | 35.43±14.62** |
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 C127 cell proliferation, reduces the Growth of Cells number of C127 cell, and this effect is dose dependent.
Claims (2)
1. Herba Boenninghauseniae cough with asthma sheet suppresses the application in breast tumor cell C127 cell proliferation medicine in preparation, described Herba Boenninghauseniae cough with asthma sheet is made up as crude drug of 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 made up 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, for subsequent use; 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, for subsequent use; 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. suppress the application in breast tumor cell C127 cell proliferation medicine according to the Herba Boenninghauseniae cough with asthma sheet described in claim l in preparation, described Herba Boenninghauseniae cough with asthma sheet is made up as crude drug of 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 made up 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, for subsequent use; 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, for subsequent use; 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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Cited By (1)
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| CN104288659A (en) * | 2014-10-16 | 2015-01-21 | 济南新起点医药科技有限公司 | Preparation method of fomes officinalis cough and asthma treating tablets and application of fomes officinalis cough and asthma treating tablets in preparation of medicine for inhibiting cell proliferation of mouse breast tumor cell C127 |
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Application publication date: 20140521 |