CN103768560A - Preparation method of Shuangjiang stomachache tablet and application of Shuangjiang stomachache tablet in preparing drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation - Google Patents
Preparation method of Shuangjiang stomachache tablet and application of Shuangjiang stomachache tablet in preparing drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and particularly provides a preparation method and application of a Shuangjiang stomachache tablet. The preparation method comprises the following steps: by using 200g of curcuma, 200g of Zingiber purpureum Rosc., 200g of delavay stephania root, 200g of Japanese sweetflag and 200g of semen integrifoliae as active pharmaceutical ingredients, carrying out microwave extraction and macroporous resin adsorption so that the curcumin content is greatly enhanced. The invention also provides application of the Shuangjiang stomachache tablet in preparing drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the preparation method of a kind of pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam and suppress the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
Background technology
Two Rhizoma Zingiberis Recens pills for curing stomach-ache standard No. WS-11105(ZD-1105)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicine taste fascicle.Made as crude drug by Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei, there is regulating QI to relieve pain, effect of regulating the stomach and sending down the abnormal ascending QI.For gastral cavilty feeling of fullness distending pain due to QI stagnating in middle-JIAO, belch acid regurgitation, chronic superficial gastritis is shown in above-mentioned patient.
In prior art, not yet have two Rhizoma Zingiberis Recens tabellae contragasteralgiams at the report that adopts microwave technology and macroporous adsorption resin technology to extract aspect extraction preparation, and Chinese medicine is directly played powder extraction, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides the preparation method of a kind of pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam.
Another object of the present invention is to provide a kind of pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam 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:
The preparation method of a kind of pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam, by Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei makes as crude drug, it is characterized in that, described preparation method is made up of the following step: get upper gomi herbs, pulverize, add 70% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, microwave extraction thing is mixed and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
In the preparation method of above-mentioned two Rhizoma Zingiberis Recens tabellae contragasteralgiams, described microwave extracting power is 700W.
In the preparation method of above-mentioned two Rhizoma Zingiberis Recens tabellae contragasteralgiams, the each extraction time of described microwave extracting is 8 minutes.
Above-mentioned two Rhizoma Zingiberis Recens tabellae contragasteralgiams suppress the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
In prior art, two Rhizoma Zingiberis Recens pills for curing stomach-ache are oral, a 1.5～3g, 3 times on the one.Two Rhizoma Zingiberis Recens pills for curing stomach-ache dosages are large.The every heavy 0.3g of two Rhizoma Zingiberis Recens tabellae contragasteralgiams that adopts the inventive method to be prepared into only needs 1-2 sheet at every turn, within 1st, takes 3 times.Under the condition with more active component, greatly reduce dose.This conclusion can be by following evidence.
The comparison of curcumin content in two Rhizoma Zingiberis Recens tabellae contragasteralgiams prepared by test one, distinct methods
1, the two Rhizoma Zingiberis Recens tabellae contragasteralgiams of instrument and reagent the present invention: press embodiment 1 method preparation, use 1000g crude drug, make 500 through extracting, every heavy 0.3g.Former pair of Rhizoma Zingiberis Recens pills for curing stomach-ache, according to WS-11105(ZD-1105)-2002 standard method preparations, in contrast.Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); ; AE240 electronic analytical balance; Curcumin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Get this product, porphyrize, (former pair of Rhizoma Zingiberis Recens pills for curing stomach-ache got 3g sample in sampling, of the present invention pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam got 0.9g), accurately weighed, put in tool plug conical flask, precision adds dehydrated alcohol 20ml, close plug, weighed weight, supersound process (power 250W, frequency 33kHz) 30 minutes, lets cool, weighed weight again, supplies the weight of less loss with dehydrated alcohol, shake up, filter, get subsequent filtrate, as need testing solution.Separately get curcumin reference substance appropriate, accurately weighed, add dehydrated alcohol and make the solution of every 1ml containing 0.12mg, product solution in contrast.Test according to thin layer chromatography (appendix VI B of Chinese Pharmacopoeia version in 2000), accurate need testing solution 2 μ l, reference substance solution 1 μ l and the 2 μ l of drawing, cross point is on same silica gel g thin-layer plate respectively, take toluene-formic acid-glacial acetic acid (30: 3: 1) as developing solvent, launch, take out, dry, scan according to thin layer chromatography (appendix VI B thin layer chromatography scanning of Chinese Pharmacopoeia version in 2000), wavelength: λ s=425nm, λ R=560nm, measures test sample trap integrated value and reference substance trap integrated value, calculate, to obtain final product.
3, result
Result shows, in the two Rhizoma Zingiberis Recens tabellae contragasteralgiams of the present invention, the content of curcumin is 3-6mg/ sheet; And the content of curcumin is 0.35mg/g in former pair of Rhizoma Zingiberis Recens pills for curing stomach-ache, every Rhizoma Wenyujin Concisum cellulose content is equivalent to 9-18 times of the every g content of former pill, and in the situation that dose reduces, curcumin content improves a lot.
Above-mentioned research shows, the two Rhizoma Zingiberis Recens tabellae contragasteralgiams that adopt preparation method of the present invention to prepare, active constituent content is far away higher than WS-11105(ZD-1105) two Rhizoma Zingiberis Recens pills for curing stomach-ache of preparing 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 Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei, by gomi herbs, pulverize, add 70% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 700W, extract 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, microwave extraction thing is mixed and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of curcumin is 5.92mg/ sheet.
Embodiment 2
Get Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei, by gomi herbs, pulverize, add 70% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extract 2 times, each 5 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, microwave extraction thing is mixed and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of curcumin is 3.66mg/ sheet.
Embodiment 3
Get Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei, by gomi herbs, pulverize, add 70% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 800W, extract 2 times, each 10 minutes, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, microwave extraction thing is mixed and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
After testing, in finished product, the content of curcumin is 4.58mg/ sheet.
Embodiment 4: two Rhizoma Zingiberis Recens tabellae contragasteralgiams suppress 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: the two Rhizoma Zingiberis Recens tabellae contragasteralgiams of the present invention: press embodiment 1 method preparation.
Medicinal liquid liquid storage: take the two Rhizoma Zingiberis Recens tabellae contragasteralgiams of 100mg, 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); NaHCO
3(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) RM-1 cell uses DMEM+10%FBS in 37 ℃, 5%CO
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%CO
2) cellar culture.
3) according to Growth of Cells situation, generally grow to 70%-70%, add two Rhizoma Zingiberis Recens tabellae contragasteralgiam 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 ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, with matched group comparison, in the time that dosage reaches 5mg/ml, to RM-1 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 pair Rhizoma Zingiberis Recens tabellae contragasteralgiam is on RM-1 cell inhibitory effect impact research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 8.71±8.24 |
| 2 | 10 | 18.77±13.12* |
| 3 | 15 | 32.49±17.05** |
| 4 | 20 | 43.56±22.28** |
Note: with matched group comparison, * P<0.01; * P<0.001.
5. experiment conclusion
Two Rhizoma Zingiberis Recens tabellae contragasteralgiams can suppress RM-1 cell proliferation, reduce the Growth of Cells number of RM-1 cell, and this effect is dose dependent.
Claims (4)
1. the preparation method of two Rhizoma Zingiberis Recens tabellae contragasteralgiams, by Rhizoma Curcumae Longae 200g, purple Rhizoma Zingiberis Recens 200g, Radix Stephaniae Epigaeae 200g, Rhizoma Acori Graminei 200g, the sub-200g of Herba Sonchi Oleracei makes as crude drug, it is characterized in that, described preparation method is made up of the following step: get upper gomi herbs, pulverize, add 70% ethanol of 5L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600-800W, extract 2 times, each 5-10 minute, combining extraction liquid, concentrated, be added on Diaion HP-10 macroporous adsorptive resins, 70% ethanol elution, collect 5 times of amount column volume eluents, decompression recycling ethanol, concentrate and be dried, obtain microwave extraction thing, microwave extraction thing is mixed and adds starch, 70% ethanol granule processed, dry, tabletting, make 500, every heavy 0.3g.
2. according to the preparation method of the two Rhizoma Zingiberis Recens tabellae contragasteralgiams described in claim l, it is characterized in that, described microwave extracting power is 700W.
3. the preparation method of according to claim 1 pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam, is characterized in that, the each extraction time of described microwave extracting is 8 minutes.
4. claimed in claim 1 pair of Rhizoma Zingiberis Recens tabellae contragasteralgiam suppresses the application in Prostate Carcinoma of Mice cell RM-1 cell proliferation medicine in preparation.
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| CN201310658935.4A CN103768560A (en) | 2013-12-06 | 2013-12-06 | Preparation method of Shuangjiang stomachache tablet and application of Shuangjiang stomachache tablet in preparing drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation |
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| CN201310658935.4A CN103768560A (en) | 2013-12-06 | 2013-12-06 | Preparation method of Shuangjiang stomachache tablet and application of Shuangjiang stomachache tablet in preparing drugs for inhibiting mouse prostate cancer cell RM-1 from cell proliferation |
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| CN102988723A (en) * | 2012-10-08 | 2013-03-27 | 李正梅 | Preparation method and application of yin tonifying and calming tablet |
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| CN102988723A (en) * | 2012-10-08 | 2013-03-27 | 李正梅 | Preparation method and application of yin tonifying and calming tablet |
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| 国家药品监督管理局: "双姜胃痛丸", 《国家药品标准（试行）颁布件》 * |
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