CN104352931A - Preparation method of fomitopsis officinalis tablet capable of treating cough and asthma and application thereof in preparing drug for inhibiting cell proliferation of mouse melanoma cell B16 - Google Patents

Preparation method of fomitopsis officinalis tablet capable of treating cough and asthma and application thereof in preparing drug for inhibiting cell proliferation of mouse melanoma cell B16 Download PDF

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Publication number
CN104352931A
CN104352931A CN201410550311.5A CN201410550311A CN104352931A CN 104352931 A CN104352931 A CN 104352931A CN 201410550311 A CN201410550311 A CN 201410550311A CN 104352931 A CN104352931 A CN 104352931A
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preparation
fomitopsis officinalis
extraction
asthma
fructificatio fomitopsis
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孙波
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Jinan Xinqidian Medical Technology Co Ltd
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Jinan Xinqidian Medical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/17—Gnetophyta, e.g. Ephedraceae (Mormon-tea family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/285—Aucklandia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/89—Cyperaceae (Sedge family)
    • A61K36/8905—Cyperus (flatsedge)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9064—Amomum, e.g. round cardamom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a preparation method of a fomitopsis officinalis tablet capable of treating cough and asthma and application of the tablet. The fomitopsis officinalis tablet capable of treating cough and asthma is prepared from 20 g of fomitopsis officinalis, 100 g of plum, 80 g of reed rhizome, 60 g of licorice, 50 g of ephedra, 30 g of elecampane, 120 g of aniseed, 30 g of rhizoma cyperi, and 20 g of cardamom as raw materials, and prepared by supercritical extraction and microwave-assisted extraction, so as to greatly increase the content of glycyrrhizic acid. The invention further provides application of the fomitopsis officinalis tablet capable of treating cough and asthma in preparing a drug for inhibiting cell proliferation of mouse melanoma cell B16.

Description

A kind of preparation method of Fructificatio Fomitopsis Officinalis cough with asthma sheet and the application in suppression B16 mouse melanoma cell line cell proliferation thereof
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of Fructificatio Fomitopsis Officinalis cough with asthma sheet and suppressing the application in B16 mouse melanoma cell line cell proliferation.
Background technology
Fructificatio Fomitopsis Officinalis Kechuan oral liquid standard No. WS-10245 (ZD-0245)-2002, is recorded in national standard for traditional Chinese medicines compilation internal medicine lung system fascicle.Be made up as crude drug of Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, there is cough-relieving, relieving asthma, the effect of eliminating the phlegm.For cold cough, cough up phlegm not well, acute/chronic bronchitis, asthma.
In prior art, Fructificatio Fomitopsis Officinalis Kechuan oral liquid is not yet had to extract the report adopting supercritical extraction and microwave extraction technology in preparation, and volatile oil is drawn, the method of water heating extraction, technique is coarse, backward, and impurity is many, causes patient's consumption very large, be inconvenient to take, had a strong impact on this product and applied clinically.
In prior art, each 10ml of Fructificatio Fomitopsis Officinalis Kechuan oral liquid, 3 times on the one.Fructificatio Fomitopsis Officinalis Kechuan oral liquid is oral liquid because of it, and taste is more bitter, is not suitable for child administration, and in addition, if seasoning needs sugaring many, uncomfortable diabetic to take, patient compliance is poor.The heavy 0.3g of the every sheet of the Fructificatio Fomitopsis Officinalis cough with asthma sheet adopting the inventive method to be prepared into, only needs 2 at every turn, within 1st, takes 3 times.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.And being prepared into tablet, patient swallows with water, and dosage is little and without bitterness, patient is easy to accept.
The comparison of glycyrrhizic acid content in Fructificatio Fomitopsis Officinalis cough with asthma sheet prepared by test one, distinct methods
L, instrument and reagent Fructificatio Fomitopsis Officinalis cough with asthma of the present invention sheet: by the preparation of embodiment 1 method, use 560g crude drug, makes 200 through extracting, the heavy 0.3g of every sheet.Former Fructificatio Fomitopsis Officinalis Kechuan oral liquid, prepares according to WS-10245 (ZD-0245)-2002 standard method.Agilent1200 high performance liquid chromatograph; METTLER AE240 electronic analytical balance; Glycyrrhizic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
Chromatographic condition and system suitability octadecyl silane are filler; Methanol-0.2mol/L Spirit of Mindererus .-glacial acetic acid (67: 33: 1) is mobile phase; Determined wavelength is 250nm.Number of theoretical plate calculates should be not less than 2000 by monoammonium glycyrrhizinate peak.
The preparation precision of reference substance solution takes monoammonium glycyrrhizinate reference substance 10mg, puts in 50ml measuring bottle, dissolves and be diluted to scale, shake up with mobile phase, obtains (containing monoammonium glycyrrhizinate reference substance 0.2mg, amount to glycyrrhizic acid is 0.1959mg to every 1ml).
Fructificatio Fomitopsis Officinalis cough with asthma sheet of the present invention is got in the preparation of Fructificatio Fomitopsis Officinalis cough with asthma sheet need testing solution of the present invention, and porphyrize, gets 0.6g, accurately weighed, put in tool plug conical flask, the 10ml that adds water makes dissolving, solution filters, precision measures 2ml filtrate, moves in evaporating dish, evaporate to dryness, residue adds the flowing mutual-assistance and dissolves, move in 10ml measuring bottle, add mobile phase to scale, to obtain final product.
The Fructificatio Fomitopsis Officinalis Kechuan oral liquid of contrast is got in the preparation of reference product need testing solution, and precision measures 2ml, moves in evaporating dish, evaporate to dryness, and residue adds the flowing mutual-assistance and dissolves, and moves in 10ml measuring bottle, adds mobile phase to scale, to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, shakes up, to obtain final product.
3, result
Result shows, in Fructificatio Fomitopsis Officinalis cough with asthma sheet of the present invention, the content of glycyrrhizic acid is 3.6-7.2mg/ sheet; And the content of glycyrrhizic acid is 0.36mg/ml in former Fructificatio Fomitopsis Officinalis Kechuan oral liquid, the glycyrrhizic acid content each serving consumption 2 is 2-4 times of original oral liquid agent content, and when dose reduces, glycyrrhizic acid content improves a lot.
Above-mentioned research shows, adopts Fructificatio Fomitopsis Officinalis cough with asthma sheet prepared by preparation method of the present invention, Fructificatio Fomitopsis Officinalis Kechuan oral liquid prepared by the method that active constituent content is recorded higher than WS-10245 (ZD-0245)-2002 standard far away.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of preparation method of Fructificatio Fomitopsis Officinalis cough with asthma sheet.
Another object of the present invention is to provide a kind of Fructificatio Fomitopsis Officinalis cough with asthma sheet to suppress the application in B16 mouse melanoma cell line cell proliferation in preparation.
The object of the invention is by following scheme realize:
A kind of preparation method of Fructificatio Fomitopsis Officinalis cough with asthma sheet, be made up as crude drug of Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, described preparation method is made up of the following step: get anisum fruit, Herba Ephedrae, the Radix Aucklandiae, Rhizoma Cyperi, Fructus Amomi Rotundus, 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 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 150-200min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 3L, 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, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 200, the heavy 0.3g of every sheet.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described CO 2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described CO 2in supercritical extraction, extracting pressure is 25MPa.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described CO 2in supercritical extraction, extraction temperature is 40 DEG C.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described CO 2cO in supercritical extraction 2flow 2ml/g crude drug min.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described CO 2in supercritical extraction, extraction time is 180min.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, described microwave extracting power is 700W.
Preferably, in the preparation method of above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet, each extraction time of described microwave extracting is 8 minutes.
Above-mentioned Fructificatio Fomitopsis Officinalis cough with asthma sheet suppresses the application in B16 mouse melanoma cell line cell proliferation in preparation.
Detailed description of the invention
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 that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, Herba speranskiae tuberculatae, Radix seu Herba Gei aleppici, Rhizoma Curcumae Longae are joined 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, temperature 40 DEG C, CO 2flow 2ml/g crude drug min, extraction time 180min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 3L, 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, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of glycyrrhizic acid is 7.25mg/ sheet.
Embodiment 2
Get Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, Herba speranskiae tuberculatae, Radix seu Herba Gei aleppici, Rhizoma Curcumae Longae are joined 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 150min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 3L, 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, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of glycyrrhizic acid is 4.52mg/ sheet.
Embodiment 3
Get Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, Herba speranskiae tuberculatae, Radix seu Herba Gei aleppici, Rhizoma Curcumae Longae are joined 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, temperature 30 DEG C, CO 2flow 1ml/g crude drug min, extraction time 200min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 3L, 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, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 200, the heavy 0.3g of every sheet.
After testing, in finished product, the content of glycyrrhizic acid is 3.68mg/ sheet.
Embodiment 4: Fructificatio Fomitopsis Officinalis cough with asthma sheet suppresses the experimentation data of B16 mouse melanoma cell line cell proliferation
1. experiment material
1.1 experiment cell strains
B16 mouse melanoma cell line cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Fructificatio Fomitopsis Officinalis cough with asthma sheet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg Fructificatio Fomitopsis Officinalis cough with asthma sheet, be dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061 Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC0 3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033 Lot.No.1088387 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: SPECTRAMAX 190); C0 2incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. 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 company in Shanghai 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 μm of 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) B16 cell DMEM+10%FBS is in 37 DEG C, 5%C0 2carry out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml 0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 10 4individual/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, culture plate put into cell culture incubator (37 DEG C, 5%C0 2) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add Fructificatio Fomitopsis Officinalis cough with asthma sheet solution, continue to cultivate 24h.
4) add 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole, as entered 200 μ l dimethyl sulfoxide, is put low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.
7) result represents with the suppression ratio of medicine to cell: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repetition 3 times.
3. statistical disposition
Adopt the correlation analysis in Microsoft Excel 2007 software and Student t to check, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to B16 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has pole significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 Fructificatio Fomitopsis Officinalis cough with asthma sheet is to B16 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 10.15±7.06
2 10 18.22±14.17*
3 15 36.95±15.84**
4 20 48.44±19.13**
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Fructificatio Fomitopsis Officinalis cough with asthma sheet of the present invention can suppress B16 cell proliferation, and reduce the Growth of Cells number of B16 cell, this effect is dose dependent.

Claims (9)

1. the preparation method of a Fructificatio Fomitopsis Officinalis cough with asthma sheet, be made up as crude drug of Fructificatio Fomitopsis Officinalis 20g, european plum 100g, Rhizoma Phragmitis 80g, Radix Glycyrrhizae 60g, Herba Ephedrae 50g, Radix Aucklandiae 30g, anisum fruit 120g, Rhizoma Cyperi 30g, Fructus Amomi Rotundus 20g, it is characterized in that, described preparation method is made up of the following step: get anisum fruit, Herba Ephedrae, the Radix Aucklandiae, Rhizoma Cyperi, Fructus Amomi Rotundus, 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 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 150-200min, obtains supercritical extract, for subsequent use; Get all the other Chinese medicines, pulverize, add 70% ethanol of 3L, 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, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrated and dry, obtain microwave extraction thing, for subsequent use; By above-mentioned supercritical extract and the mixing of microwave extraction thing, add starch, 70% ethanol granule, dry, tabletting, makes 200, the heavy 0.3g of every sheet.
2. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described CO 2in supercritical extraction, entrainer accounts for the percent by volume of total extractant is 5%.
3. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described CO 2in supercritical extraction, extracting pressure is 25 MPa.
4. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described CO 2in supercritical extraction, extraction temperature is 40 DEG C.
5. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described CO 2cO in supercritical extraction 2flow 2ml/g crude drug min.
6. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described CO 2in supercritical extraction, extraction time is 180min.
7. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, described microwave extracting power is 700W.
8. the preparation method of the Fructificatio Fomitopsis Officinalis cough with asthma sheet according to claim l, is characterized in that, each extraction time of described microwave extracting is 8 minutes.
9. the Fructificatio Fomitopsis Officinalis cough with asthma sheet described in claim l suppresses the application in B16 mouse melanoma cell line cell proliferation in preparation.
CN201410550311.5A 2014-10-16 2014-10-16 Preparation method of fomitopsis officinalis tablet capable of treating cough and asthma and application thereof in preparing drug for inhibiting cell proliferation of mouse melanoma cell B16 Pending CN104352931A (en)

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Citations (1)

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CN103800499A (en) * 2013-12-10 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 melanoma cell B16 from cell proliferation

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