CN104161852A - Preparation method for nine-composition hemorrhoid tablet and application thereof to medicines inhibiting cell proliferation of prostate cancer cell RM-1 - Google Patents
Preparation method for nine-composition hemorrhoid tablet and application thereof to medicines inhibiting cell proliferation of prostate cancer cell RM-1 Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and concretely relates to a preparation method for a nine-composition hemorrhoid tablet and application. According to the preparation method for the nine-composition hemorrhoid tablet, 400 g of rubus hirsutus, 150 g of rhizoma polygoni cuspidati, 200 g of radix sanguisorbae, 100 g of herba visci, 100 g of ficus carica leaf, 50 g of flos chrysanthemi, 20 g of egg white, 150 g of rhizoma coptidis and 50 g of radix et rhizoma rhei are employed as bulk drugs, and supercritical extraction and microwave extraction are employed, so that the nine-composition hemorrhoid tablet is substantially improved in emodin content. The invention also provides application of the nine-composition hemorrhoid tablet to prepare medicines inhibiting cell proliferation of mouse prostate cancer cell RM-1.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method and application in suppressing Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine thereof for the treatment of piles with 9-medicinal herbs sheet.
Background technology
Capsule for treating piles with 9-medicinal herbs standard No. WS-10189 (ZD-0189)-2002, is recorded in national standard for traditional Chinese medicines compilation surgery gynecological fascicle.By Herba Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, as crude drug, made, there is heat-clearing and toxic substances removing, dampness detumescence, the effect of cooling blood for hemostasis.。For Internal hemorrhoid hemorrhage due to damp-heat accumulation, external hemorrhoid welling and pain.
In prior art, not yet there is capsule for treating piles with 9-medicinal herbs adopting the report of supercritical and microwave technology aspect extraction preparation, and directly beat powder, the method that decocting boils, technique is coarse, backward, and impurity is many, cause patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
In prior art, the each 5-6 grain of capsule for treating piles with 9-medicinal herbs, 3 times on the one.Capsule for treating piles with 9-medicinal herbs dosage is large, and the every heavy 0.3g for the treatment of piles with 9-medicinal herbs sheet that adopts the inventive method to be prepared into only needs 3 at every turn, within 1st, takes 3 times.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 emodin content in treating piles with 9-medicinal herbs sheet prepared by test one, distinct methods
L, instrument and reagent treating piles with 9-medicinal herbs sheet of the present invention: press embodiment 1 method preparation, use 1220g crude drug, make 500 through extracting, every heavy 0.3g.Former capsule for treating piles with 9-medicinal herbs, prepares according to WS-10189 (ZD-0189)-2002 standard method.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Emodin 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 octadecylsilane chemically bonded silica are filler; Methanol-0.1% phosphoric acid solution (80:20) is mobile phase; Detection wavelength is 288nm.Number of theoretical plate calculates and should be not less than 5000 by emodin peak.
It is appropriate that the preparation precision of reference substance solution takes emodin reference substance, adds methanol and make every 1ml containing the solution of 0.05mg, obtains.
Treating piles with 9-medicinal herbs sheet of the present invention is got in the preparation of need testing solution, and porphyrize, gets 0.5g, accurately weighed, put in tool plug conical flask, add methanol 20ml, supersound process 1 hour, lets cool, filter residue methanol wash 2 times, each 10ml, merging filtrate and washing liquid, evaporate to dryness, residue adds dilute hydrochloric acid 10ml to be made to dissolve, add again chloroform 20ml, put (70 ℃) in water-bath and be hydrolyzed 30 minutes, cooling, in dislocation separatory funnel, with a small amount of chloroform washing container, be incorporated in separatory funnel; Minute get chloroform layer, chloroform washing is 2 times for acid solution, each 10ml, combined chloroform liquid, with anhydrous sodium sulfate, dewater in right amount, fling to chloroform, residue makes to dissolve and be transferred in 10ml measuring bottle with methanol in right amount, adds methanol to scale, shake up, with microporous filter membrane (0.45 m), filter, get subsequent filtrate, obtain.
The capsule for treating piles with 9-medicinal herbs of this contrast is got in the preparation of reference product need testing solution, and porphyrize mixes, and gets 1g, accurately weighed, put in tool plug conical flask, add methanol 20ml, supersound process 1 hour, let cool, filter, residue methanol wash 2 times, each 10ml, merging filtrate and washing liquid, evaporate to dryness, residue adds dilute hydrochloric acid 10ml to be made to dissolve, add again chloroform 20ml, put (70 ℃) in water-bath and be hydrolyzed 30 minutes, cooling, in dislocation separatory funnel, with a small amount of chloroform washing container, be incorporated in separatory funnel; Minute get chloroform layer, chloroform washing is 2 times for acid solution, each 10ml, combined chloroform liquid, with anhydrous sodium sulfate, dewater in right amount, fling to chloroform, residue makes to dissolve and be transferred in 10ml measuring bottle with methanol in right amount, adds methanol to scale, shake up, with microporous filter membrane (0.45 m), filter, get subsequent filtrate, obtain.
Algoscopy is accurate reference substance solution and each 5 l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.3, result
Result shows, in treating piles with 9-medicinal herbs sheet of the present invention, the content of emodin is 0.65-1.22mg/ sheet; And the content of emodin is 0.15mg/ grain in former capsule for treating piles with 9-medicinal herbs, each serving the emodin content of 3 of consumptions be original capsule content 2-4 doubly, in the situation that dose reduces, emodin content improves a lot.
Above-mentioned research shows, the treating piles with 9-medicinal herbs sheet that adopts preparation method of the present invention to prepare, capsule for treating piles with 9-medicinal herbs prepared by the method that active constituent content is recorded higher than WS-10189 (ZD-0189)-2002 standard far away.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method for the treatment of piles with 9-medicinal herbs sheet.
Another object of the present invention is to provide a kind for the treatment of piles with 9-medicinal herbs sheet to suppress the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
The object of the invention is to realize by following scheme:
A kind of preparation method for the treatment of piles with 9-medicinal herbs sheet, by Herba Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, as crude drug, made, described preparation method is comprised of the following step: get Herba Rubi parvifolii, Rhizoma Polygoni Cuspidati, Liu Jisheng, 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 60% 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 Dl01 macroporous adsorptive resins, 60% 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 500, every heavy 0.3g.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described CO
2in supercritical extraction, extracting pressure is 25 MPa.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described CO
2in supercritical extraction, extraction temperature is 40 ℃.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described CO
2in supercritical extraction, extraction time is 200min.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, described microwave extracting power is 700W.
In the preparation method of above-mentioned treating piles with 9-medicinal herbs sheet, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned treating piles with 9-medicinal herbs sheet suppresses the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
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 Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, Herba Rubi parvifolii, Rhizoma Polygoni Cuspidati, Liu Jisheng 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 60% 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 Dl01 macroporous adsorptive resins, 60% 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 500, every heavy 0.3g.
After testing, in finished product, the content of emodin is 1.22mg/ sheet.
Embodiment 2
Get Herba Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, Herba Rubi parvifolii, Rhizoma Polygoni Cuspidati, Liu Jisheng 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 60% 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 Dl01 macroporous adsorptive resins, 60% 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 500, every heavy 0.3g.
After testing, in finished product, the content of emodin is 0.87mg/ sheet.
Embodiment 3
Get Herba Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, Herba Rubi parvifolii, Rhizoma Polygoni Cuspidati, Liu Jisheng 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 60% 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 Dl01 macroporous adsorptive resins, 60% 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 500, every heavy 0.3g.
After testing, in finished product, the content of emodin is 0.65mg/ sheet.
Embodiment 4: treating piles with 9-medicinal herbs sheet suppresses the experimentation data of Prostate Carcinoma of Mice cell RM-1 cell proliferation
1. experiment material
1.1 experiment cell strains
Prostate Carcinoma of Mice cell RM-1 cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: treating piles with 9-medicinal herbs sheet of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take 100mg treating piles with 9-medicinal herbs sheet, be dissolved in 5ml dehydrated alcohol, 0.2 m filter filters, 500 ldoff pipe subpackages ,-20 ℃ of storages, 0.2 m filter filters dehydrated alcohol in order to the use of 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 Cat.No.11810-033 Lot.No. of Shanghai Jiu Yi chemical reagent company limited 1088387); 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: SPECTRAMAX 190); 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/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 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) RM-1 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 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 treating piles with 9-medicinal herbs sheet solution, continue to cultivate 24h.
4) after 24h, add 20 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 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 Excel 2007 softwares, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to RM-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 treating piles with 9-medicinal herbs sheet is on RM-1 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 9.54±6.98 |
| 2 | 10 | 17.77±8.64* |
| 3 | 15 | 29.46±11.65** |
| 4 | 20 | 38.96±14.36** |
Note: with matched group comparison, * P<O.01; * P<0.001.
5. experiment conclusion
Treating piles with 9-medicinal herbs sheet of the present invention can suppress RM-1 cell proliferation, reduces the Growth of Cells number of RM-1 cell, and this effect is dose dependent.
Claims (9)
1. the preparation method of a treating piles with 9-medicinal herbs sheet, by Herba Rubi parvifolii 400g, Rhizoma Polygoni Cuspidati 150g, Radix Sanguisorbae 200g, Liu Jisheng 100g, common fig leaf juice 100g, Flos Chrysanthemi 50g, Ovum Gallus domesticus album 20g, Rhizoma Coptidis 150g, Radix Et Rhizoma Rhei 50g, as crude drug, made, it is characterized in that, described preparation method is comprised of the following step: get Herba Rubi parvifolii, Rhizoma Polygoni Cuspidati, Liu Jisheng, 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 60% 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 Dl01 macroporous adsorptive resins, 60% 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 500, every heavy 0.3g.
2. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, to account for the percent by volume of total extractant be 5% to entrainer.
3. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extracting pressure is 25 MPa.
4. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction temperature is 40 ℃.
5. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that described CO
2cO in supercritical extraction
2flow 2ml/g crude drug min.
6. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that described CO
2in supercritical extraction, extraction time is 200min.
7. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that, described microwave extracting power is 700W.
8. according to the preparation method of the treating piles with 9-medicinal herbs sheet described in claim l, it is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
9. the treating piles with 9-medicinal herbs sheet described in claim l suppresses the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104116810A (en) * | 2014-07-31 | 2014-10-29 | 济南爱思医药科技有限公司 | Preparation method of Jiuwei haemorrhoids tablet and application of Jiuwei haemorrhoids tablet to medicine for inhibiting cell proliferation of lymphoma cell YAC-1 |
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| CN103800498A (en) * | 2013-12-06 | 2014-05-21 | 济南新起点医药科技有限公司 | Preparation method of Qingmei cold tablet and application of Qingmei cold tablet in drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103800498A (en) * | 2013-12-06 | 2014-05-21 | 济南新起点医药科技有限公司 | Preparation method of Qingmei cold tablet and application of Qingmei cold tablet in drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104116810A (en) * | 2014-07-31 | 2014-10-29 | 济南爱思医药科技有限公司 | Preparation method of Jiuwei haemorrhoids tablet and application of Jiuwei haemorrhoids tablet to medicine for inhibiting cell proliferation of lymphoma cell YAC-1 |
| CN104116810B (en) * | 2014-07-31 | 2016-03-23 | 济南爱思医药科技有限公司 | A kind of preparation method for the treatment of piles with 9-medicinal herbs sheet and the application in suppression lymphoma cell YAC-1 cell proliferation thereof |
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