CN102988480B - Method for preparing bile and patchouli rhinitis tablets and application - Google Patents
Method for preparing bile and patchouli rhinitis tablets and application Download PDFInfo
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- CN102988480B CN102988480B CN201210378476.XA CN201210378476A CN102988480B CN 102988480 B CN102988480 B CN 102988480B CN 201210378476 A CN201210378476 A CN 201210378476A CN 102988480 B CN102988480 B CN 102988480B
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- 206010039083 rhinitis Diseases 0.000 title claims abstract description 40
- 210000000941 bile Anatomy 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title abstract description 11
- 235000011751 Pogostemon cablin Nutrition 0.000 title abstract 4
- 241000222666 Boerhavia diffusa Species 0.000 title 1
- 238000002360 preparation method Methods 0.000 claims abstract description 25
- 239000003814 drug Substances 0.000 claims abstract description 24
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000000874 microwave-assisted extraction Methods 0.000 claims abstract description 11
- 238000000194 supercritical-fluid extraction Methods 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 55
- 210000000232 gallbladder Anatomy 0.000 claims description 33
- 241000628997 Flos Species 0.000 claims description 22
- 239000000284 extract Substances 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 17
- 229940079593 drug Drugs 0.000 claims description 13
- 239000009670 cang er zi wan Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- 230000004663 cell proliferation Effects 0.000 claims description 8
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 7
- 201000005202 lung cancer Diseases 0.000 claims description 7
- 208000020816 lung neoplasm Diseases 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 5
- 230000000274 adsorptive effect Effects 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 5
- 230000006837 decompression Effects 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000008187 granular material Substances 0.000 claims description 5
- 238000004064 recycling Methods 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 239000011347 resin Substances 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 240000002505 Pogostemon cablin Species 0.000 abstract 3
- 241001506766 Xanthium Species 0.000 abstract 2
- 235000013399 edible fruits Nutrition 0.000 abstract 2
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 abstract 1
- 241000213006 Angelica dahurica Species 0.000 abstract 1
- 241000205585 Aquilegia canadensis Species 0.000 abstract 1
- 241001633683 Centipeda <firmicute> Species 0.000 abstract 1
- 244000035851 Chrysanthemum leucanthemum Species 0.000 abstract 1
- 235000008495 Chrysanthemum leucanthemum Nutrition 0.000 abstract 1
- 206010023774 Large cell lung cancer Diseases 0.000 abstract 1
- 241000951473 Schizonepeta Species 0.000 abstract 1
- 201000009546 lung large cell carcinoma Diseases 0.000 abstract 1
- 229940041616 menthol Drugs 0.000 abstract 1
- 229960004756 ethanol Drugs 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000013558 reference substance Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 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
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010039085 Rhinitis allergic Diseases 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 201000010105 allergic rhinitis Diseases 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 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
- 238000004113 cell culture Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 201000009151 chronic rhinitis Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012738 dissolution medium Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
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- 239000000945 filler Substances 0.000 description 1
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- 238000004900 laundering Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- JWHHANVGNNWIRI-UHFFFAOYSA-N methanol phosphoric acid hydrate Chemical compound O.OC.OP(O)(O)=O JWHHANVGNNWIRI-UHFFFAOYSA-N 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 201000009890 sinusitis Diseases 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a method for preparing bile and patchouli rhinitis tablets. The rhinitis tablets are prepared from 15g of porcine bile paste, 560g of patchouli, 560g of dahurian angelica, 450g of cocklebur fruit, 375g of centipeda, 250g of schizonepeta, 250g of honeysuckle, 250g of wild chrysanthemum and 1.5g of menthol serving as raw medicaments by adopting supercritical extraction and microwave extraction, so that the cocklebur fruit content is greatly improved. The invention also provides application of the bile and patchouli rhinitis tablets in preparation of a medicament for inhibiting propagation of human large cell lung cancer NC1-H460 cells.
Description
Technical field
The present invention relates to Chinese medicine preparation technical field, be specifically related to preparation method and the application of the fragrant rhinitis tablet of a kind of gallbladder.
Background technology
The fragrant rhinitis tablet of gallbladder is recorded in Ministry of Public Health standard WS3-B-0914-91, by porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g, as crude drug, made, energy antiinflammatory heat clearing away, expelling wind and cold, understand things pain-stopping.For chronic simple rhinitis, allergic rhinitis.Acute and chronic rhinitis nasal sinusitis.
In prior art, not yet there is the fragrant rhinitis tablet of gallbladder to adopt the report of supercritical and microwave technology extracting aspect preparation, and adopt the method that powder and decocting boil of beating, 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
Goal of the invention: in order to address the above problem, the object of the present invention is to provide the preparation method of the fragrant rhinitis tablet of a kind of gallbladder.
Another object of the present invention is to provide the fragrant rhinitis tablet of a kind of gallbladder to suppress the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation.
Technical scheme: the object of the invention is to realize by following scheme:
The preparation method of the fragrant rhinitis tablet of a kind of gallbladder, by porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g makes as crude drug, described method is comprised of the following step: get Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join in CO2 supercritical extraction device, ethanol is as entrainer, the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 ℃, CO2 flow 1-3m1/g crude drug min, extraction time 150-180min, obtain supercritical extract, standby, get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400-600W, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 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 porcine bile paste, Mentholum, starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
The preparation method of the fragrant rhinitis tablet of above-mentioned a kind of gallbladder, described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
The preparation method of the fragrant rhinitis tablet of above-mentioned a kind of gallbladder, described microwave extracting power 500W extracts 6 minutes at every turn.
The preparation method of the fragrant rhinitis tablet of above-mentioned a kind of gallbladder, described CO
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
The fragrant rhinitis tablet of above-mentioned gallbladder suppresses the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation.
In prior art, every 0.5g of the fragrant rhinitis tablet of gallbladder, each 4,3 times on the one, the every 0.5g of the fragrant rhinitis tablet of gallbladder that adopts the present invention to be prepared into only needs 2 at every turn, within 1st, takes 3 times, has greatly reduced dose having under the condition of more active component.This conclusion can be by following evidence.
The comparison of Herba Xanthii spit of fland content in the fragrant rhinitis tablet of gallbladder prepared by test one, distinct methods
1, the fragrant rhinitis tablet of instrument and reagent gallbladder of the present invention: press embodiment 3 method preparations, use 2240g crude drug, make 1000 through extracting, every heavy 0.5g.The fragrant rhinitis tablet of former gallbladder, by the preparation of ministry standard method, is used 2240g crude drug, through extracting, makes 1000, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; METTLERAE240 electronic analytical balance; Herba Xanthii spit of fland reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica, be filler; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; Detection wavelength is 280nm.Number of theoretical plate calculates by Herba Xanthii Ting Feng, should be not less than 3000.
The preparation of reference substance solution: precision takes at 60 ℃ of drying under reduced pressure Herba Xanthii spit of fland reference substance of 4 hours appropriate, adds methanol and makes every 1ml containing the solution of 18 μ g, obtains.
The preparation of need testing solution: get the fragrant rhinitis tablet of the fragrant rhinitis tablet of gallbladder of the present invention and former gallbladder, porphyrize, mixes, and gets 1g, accurately weighed, and precision adds 70% ethanol 20ml, close plug, supersound process 10 minutes, centrifugal, get supernatant, obtain.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and injection liquid chromatography, measures, and obtains.
3, result
Result shows, in the fragrant rhinitis tablet of gallbladder of the present invention, the content in Herba Xanthii spit of fland is 1.67mg/ sheet; And the content in Herba Xanthii spit of fland is 0.38mg/ sheet in the fragrant rhinitis tablet of former gallbladder, in the situation that dose reduces, Herba Xanthii spit of fland content improves a lot.
Above-mentioned research shows, the fragrant rhinitis tablet of gallbladder that adopts the present invention to prepare, and active constituent content is higher than the fragrant rhinitis tablet of the standby gallbladder of ministry standard legal system.
The specific embodiment
Form by the following examples, foregoing of the present invention is described in further detail again, 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 porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g, by Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join in CO2 supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, 30 ℃ of temperature, CO2 flow 1m1/g crude drug min, extraction time 150min, obtain supercritical extract, standby; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400W, extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on D101 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 porcine bile paste, Mentholum, starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content in Herba Xanthii spit of fland is 1.66mg/ sheet.
Embodiment 2
Get porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g, by Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 30MPa, temperature 50 C, CO
2flow 3m1/g crude drug min, extraction time 180min, obtains supercritical extract, standby; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 600W, extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on D101 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 porcine bile paste, Mentholum, starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content in Herba Xanthii spit of fland is 1.73mg/ sheet.
Embodiment 3
Get porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g, by Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, 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 20MPa, 40 ℃ of temperature, CO
2flow 2m1/g crude drug min, extraction time 160min, obtains supercritical extract, standby; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 500W, extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on D101 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 porcine bile paste, Mentholum, starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
After testing, in finished product, the content in Herba Xanthii spit of fland is 1.67mg/ sheet.
Embodiment 4: the fragrant rhinitis tablet of gallbladder suppresses the experimentation data of NCI-H460 cell proliferation
1 experiment material
1.1 experiment cell strains
The cell lung cancer NCI-H460 of National People's Congress cell, Nanjing Zhengkuan Pharmaceutical Technology Co., Ltd.'s laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the fragrant rhinitis tablet of gallbladder of the present invention: press embodiment 3 method preparations.
Medicinal liquid liquid storage: take the fragrant rhinitis tablet of 100mg gallbladder, 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
The Cat.No.12100-061 Lot.No.758137 of DMEM(GIBCO company); Hyclone (Lot.No.100419 of Tian Hang bio tech ltd, Zhejiang); The Cat.No.11810-033 Lot.No.1088387 of NaHCO3(Shanghai Jiu Yi chemical reagent company limited); Trypsin(AMRESCO company lot number: 2010/04); EDTA(AMRESCO company lot number: 2009/10); Penicillin G Sodium Salt(AMRESCO company lot number: 2010242); Streptomycin Sulfate(AMRESCO company lot number: 2010382); Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182); MTT (Biosharp lot number: 0793); The autogamy of PBS(laboratory);
1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (U.S. MD company model: SPECTRAMAX 190); CO2 incubator (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. Spring 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 μ m filter (MILLIPORE model: SLGP033RB); 10cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2 experimental techniques
1) NCI-H460 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, 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 3 * 104/ml of concentration of cell suspension.
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%CO2) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add the fragrant rhinitis tablet solution of gallbladder, 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 every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, 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 dispositions
Adopt correlation analysis and Student t check in Microsoft Excel 2003 softwares, data represent with mean ± S.D..
4 experimental results
Statistical result showed after mtt assay experiment, with matched group comparison, when dosage reaches 5mg/ml, to NCI-H460 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.
The fragrant rhinitis tablet of table 1 gallbladder is on NCI-H460 cell inhibitory effect impact research (X ± SD)
Note: with matched group comparison, * P<0.01; * P<0.001
5 experiment conclusion
The fragrant rhinitis tablet of gallbladder can suppress NCI-H460 cell proliferation, reduces the Growth of Cells number of NCI-H460 cell, and this effect is dose dependent.
Claims (4)
1. the fragrant rhinitis tablet of gallbladder suppresses the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation, it is characterized in that the fragrant rhinitis tablet of a kind of gallbladder made as crude drug by porcine bile paste 15g, Herba Pogostemonis 560g, Radix Angelicae Dahuricae 560g, Fructus Xanthii 450g, Herba Centipedae 375g, Herba Schizonepetae 250g, Flos Lonicerae 250g, Flos Chrysanthemi Indici 250g, Mentholum 1.5g, preparation method is comprised of the following step: get Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, 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 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, standby; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in microwave extracting apparatus and carry out microwave extracting, extraction power 400-600W, extracts 2 times, each 4-8 minute, combining extraction liquid, concentrated, be added on D101 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 porcine bile paste, Mentholum, starch, 70% ethanol granule processed, dry, tabletting, makes 1000, every heavy 0.5g.
2. the fragrant rhinitis tablet of a kind of gallbladder suppresses the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. the fragrant rhinitis tablet of a kind of gallbladder suppresses the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation according to claim 1, it is characterized in that the power of microwave extracting described in preparation method 500W, extracts 6 minutes at every turn.
4. the fragrant rhinitis tablet of a kind of gallbladder suppresses the application in the cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine in preparation according to claim 1, it is characterized in that CO described in preparation method
2the extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2flow 2ml/g crude drug min, extraction time 160min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210378476.XA CN102988480B (en) | 2012-10-08 | 2012-10-08 | Method for preparing bile and patchouli rhinitis tablets and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210378476.XA CN102988480B (en) | 2012-10-08 | 2012-10-08 | Method for preparing bile and patchouli rhinitis tablets and application |
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| CN103623052A (en) * | 2013-09-30 | 2014-03-12 | 王洪珍 | Novel method for preparing danxiang (Chinese character) rhinitis tablet |
| CN103566023B (en) * | 2013-10-29 | 2015-11-18 | 山东省立医院 | A kind of preparation method of toothache Pianomide and application |
| CN103705683A (en) * | 2013-12-22 | 2014-04-09 | 青岛琴诚医药技术有限公司 | Preparation method and application of Sixiao pills |
| CN104257752B (en) * | 2014-10-09 | 2018-03-20 | 成都市康飞药业有限公司 | A kind of medical composition and its use for treating rhinitis |
| CN117599012A (en) * | 2023-12-08 | 2024-02-27 | 广西迪泰制药股份有限公司 | Preparation method of nose comfort tablet |
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| CN1803162A (en) * | 2005-01-12 | 2006-07-19 | 佛山德众药业有限公司 | Rhinological disease-treating pharmaceutical compositions and its preparing method |
| CN101579186B (en) * | 2009-06-01 | 2010-11-03 | 赵青菊 | Pharmaceutical rhinitis pillow for external use and treating wind-heat rhinitis |
| CN102258574B (en) * | 2011-07-13 | 2012-07-25 | 山东省立医院 | Chinese medicinal preparation for treating allergic rhinitis |
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