CN102988480A - 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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- CN102988480A CN102988480A CN201210378476XA CN201210378476A CN102988480A CN 102988480 A CN102988480 A CN 102988480A CN 201210378476X A CN201210378476X A CN 201210378476XA CN 201210378476 A CN201210378476 A CN 201210378476A CN 102988480 A CN102988480 A CN 102988480A
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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 claims abstract description 12
- 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 23
- 239000003814 drug Substances 0.000 claims abstract description 21
- 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
- 238000000605 extraction Methods 0.000 claims description 19
- 239000000284 extract Substances 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
- 238000001035 drying Methods 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 5
- 230000000274 adsorptive effect Effects 0.000 claims description 5
- 230000004663 cell proliferation Effects 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
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 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
- 239000012467 final product Substances 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
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000005303 weighing Methods 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
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- 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
- 238000002156 mixing Methods 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
- 238000012856 packing Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 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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- Medicines Containing Plant Substances (AREA)
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 the 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, 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, energy antiinflammatory heat clearing away, expelling wind and cold, understand things pain-stopping.Be used for chronic simple rhinitis, allergic rhinitis.The acute and chronic rhinitis nasal sinusitis.
In the prior art, not yet there is the fragrant rhinitis tablet of gallbladder to adopt the report of supercritical and microwave technology aspect the preparation extracting, and adopts 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 used 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 application of the fragrant rhinitis tablet of a kind of gallbladder in the preparation inhibition cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine.
Technical scheme: the objective 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 the 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 gets supercritical extract, and is for subsequent use; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in the 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 the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add porcine bile paste, Mentholum, starch, 70% ethanol granule processed, drying, tabletting is made 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 the 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 application of the fragrant rhinitis tablet of above-mentioned gallbladder in the preparation inhibition cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine.
In the 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, takes 3 times in 1st, 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 the gallbladder of test one, distinct methods preparation
1, the fragrant rhinitis tablet of instrument and reagent gallbladder of the present invention: press the preparation of embodiment 3 methods, use the 2240g crude drug, make 1000 through extraction, every heavy 0.5g.The fragrant rhinitis tablet of former gallbladder by the preparation of ministry standard method, uses the 2240g crude drug, makes 1000 through extraction, every heavy 0.5g.Agilent 1200 high performance liquid chromatographs; The 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: be filler with octadecylsilane chemically bonded silica; Methanol-water-phosphoric acid (40:60:0.2) is mobile phase; The 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 by weighing at 4 hours Herba Xanthii spit of fland reference substance of 60 ℃ of drying under reduced pressure an amount of, adds methanol and makes the solution that every 1ml contains 18 μ g, and get final product.
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, mixing is got 1g, and is accurately weighed, the accurate 70% ethanol 20ml that adds, close plug, supersound process 10 minutes, centrifugal, get supernatant, and get final product.
Algoscopy is accurate reference substance solution and each 20 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and be get final product.
3, result
The result shows, the content in Herba Xanthii spit of fland is the 1.67mg/ sheet in the fragrant rhinitis tablet of gallbladder of the present invention; And the content in Herba Xanthii spit of fland is the 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 studies show that, the fragrant rhinitis tablet of the gallbladder that adopts the present invention to prepare, active constituent content are 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 that the scope of the above-mentioned theme of the present invention only limits to following example, all technology that realizes 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, with Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join in the 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, get supercritical extract, for subsequent use; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 400W extracts 2 times, each 4 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add porcine bile paste, Mentholum, starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content in Herba Xanthii spit of fland is the 1.66mg/ sheet in the finished product.
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, with Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 600W extracts 2 times, each 8 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add porcine bile paste, Mentholum, starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content in Herba Xanthii spit of fland is the 1.73mg/ sheet in the finished product.
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, with Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in the microwave extracting apparatus and carry out microwave extracting, extraction power 500W extracts 2 times, each 6 minutes, combining extraction liquid, concentrated, be added on the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add porcine bile paste, Mentholum, starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
After testing, the content in Herba Xanthii spit of fland is the 1.67mg/ sheet in the finished product.
Embodiment 4: the fragrant rhinitis tablet of gallbladder suppresses the experimentation data of NCI-H460 cell proliferation
1 experiment material
1.1 experiment cell strain
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 the preparation of embodiment 3 methods.
The medicinal liquid liquid storage: take by weighing the fragrant rhinitis tablet of 100mg gallbladder, be dissolved in the 5ml dehydrated alcohol, 0.2 μ m filter filters, and 500 μ ldoff manage packing ,-20 ℃ of storages, and 0.2 μ m filter filters dehydrated alcohol in order to the usefulness of matched group simultaneously.
1.3 experiment reagent
The Cat.No.12100-061 Lot.No.758137 of DMEM(GIBCO company); Hyclone (Hangzhoupro, sky, Zhejiang bio tech ltd Lot.No.100419); NaHCO3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033 Lot.No.1088387 of a specified duration); 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); As seen-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 makes 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 (the accurate experimental facilities in Shanghai 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) the NCI-H460 cell carries out cellar culture (10cm culture dish) with DMEM+10%FBS in 37 ℃, 5%CO2, when Growth of Cells during to logarithmic (log) phase, collecting cell discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, behind 37 ℃ of digestion 2min, to wherein adding 5ml complete medium neutralization reaction, behind the piping and druming cell it is changed in the centrifuge tube, the centrifugal 5min of 1000rpm adjusts 3 * 104/ml of concentration of cell suspension.
2) the 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 the Growth of Cells situation, generally grow to 50%-70%, add the fragrant rhinitis tablet solution of gallbladder, continue to cultivate 24h.
4) add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) behind the 24h, continue to cultivate 4h.
5) the buckle method is removed supernatant behind the 4h, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on the shaking table, 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) is set 6 multiple holes for every group.
7) result represents with the suppression ratio of medicine to cell:
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 the mtt assay experiment, compare with matched group, 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), and utmost point significant difference (P＜0.001) is arranged 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)
Annotate: compare * P＜0.01 with matched group; * P＜0.001
5 experiment conclusion
The fragrant rhinitis tablet of gallbladder can suppress the NCI-H460 cell proliferation, reduces the Growth of Cells number of NCI-H460 cell, and this effect is dose dependent.
Claims (5)
1. the preparation method of the fragrant rhinitis tablet of a 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, it is characterized in that described method is comprised of the following step: get Herba Pogostemonis, Herba Schizonepetae, Flos Lonicerae, Flos Chrysanthemi Indici, join CO
2In the 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 gets supercritical extract, and is for subsequent use; Get the Radix Angelicae Dahuricae, Fructus Xanthii, Herba Centipedae, pulverize, add 70% ethanol of 2L, drop in the 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 the D101 macroporous adsorptive resins, 50% ethanol elution is collected 5 times of amount column volume eluents, decompression recycling ethanol, concentrated and dry, get the microwave extraction thing, for subsequent use; Above-mentioned supercritical extract and microwave extraction thing are mixed, add porcine bile paste, Mentholum, starch, 70% ethanol granule processed, drying, tabletting is made 1000, every heavy 0.5g.
2. the preparation method of the fragrant rhinitis tablet of described a kind of gallbladder according to claim 1 is characterized in that described CO
2The percent by volume that the supercritical extraction entrainer accounts for total extractant is 5%.
3. the preparation method of the fragrant rhinitis tablet of described a kind of gallbladder according to claim 1 is characterized in that described microwave extracting power 500W, extracts 6 minutes at every turn.
4. the preparation method of the fragrant rhinitis tablet of described a kind of gallbladder according to claim 1 is characterized in that described CO
2The extracting pressure 20MPa of supercritical extraction, 40 ℃ of temperature, CO
2Flow 2ml/g crude drug min, extraction time 160min.
5. the according to claim 1 application of the fragrant rhinitis tablet of described a kind of gallbladder in the preparation inhibition cell lung cancer NCI-H460 of National People's Congress cell proliferation medicine.
Priority Applications (1)
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| CN103623052A (en) * | 2013-09-30 | 2014-03-12 | 王洪珍 | Novel method for preparing danxiang (Chinese character) rhinitis tablet |
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| 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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