CN105726960A - Application of pseudobulb-of-corymbose-coelogyne cough and asthma tablets to preparation of medicine for restraining human osteosarcoma cell MG-63 multiplication - Google Patents

Application of pseudobulb-of-corymbose-coelogyne cough and asthma tablets to preparation of medicine for restraining human osteosarcoma cell MG-63 multiplication Download PDF

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CN105726960A
CN105726960A CN201610088722.6A CN201610088722A CN105726960A CN 105726960 A CN105726960 A CN 105726960A CN 201610088722 A CN201610088722 A CN 201610088722A CN 105726960 A CN105726960 A CN 105726960A
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cough
green
rhizoma
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asthma
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刘芳
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A61K36/704—Polygonum, e.g. knotweed
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
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    • 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

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Abstract

The invention belongs to the technical field of traditional Chinese medicine and particularly relates to application of pseudobulb-of-corymbose-coelogyne cough and asthma tablets to preparation of medicine for restraining human osteosarcoma cell MG-63 multiplication and a preparation method of the pseudobulb-of-corymbose-coelogyne cough and asthma tablets. The pseudobulb-of-corymbose-coelogyne cough and asthma tablets are prepared from bulk drugs including 200 g of pseudobulb of corymbose coelogyne, 200 g of paederia scandens, 100 g of Chinese mahonia stems, 100 g of marsdenia tenacissima, 100 g of rhizoma bletillae, 100 g of rhizoma polygoni cuspidate, 100 g of garden balsam stems and 100 g of rhizoma polygonati. The tablets are prepared through supercritical extraction, and the content of emodin is greatly increased.

Description

Green and cough with asthma sheet suppresses OS-732 cells MG-63 cell proliferation in preparation In application
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of green and cough with asthma sheet at preparation suppression OS-732 cells Application in MG-63 cell proliferation and the green and preparation method of cough with asthma sheet.
Background technology
Green and cough with asthma particulate level WS-10616(ZD-0616)-2002, it is recorded in country's standard for traditional Chinese medicines compilation internal medicine Lung system (two) fascicle.By little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g make as crude drug, have effect of nourishing YIN and moistening the lung, heat-clearing and toxic substances removing, removing stasis to stop bleeding, are used for The heat cough that causes of dry criminal's lung, hectic fever, the disease such as night sweat.
In prior art, not yet there is green and cough with asthma sheet at preparation suppression people's OS-732 cells MG-63 cell proliferation The report of the application in medicine, also there are no the green and report of cough with asthma sheet extraction preparation aspect employing supercritical extraction, and traditional Soak by water, the method for alcohol reflux, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, it has not been convenient to take, sternly Heavily have impact on this product to apply clinically.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of green and cough with asthma sheet at preparation suppression OS-732 cells Application in MG-63 cell proliferation.
A kind of green and preparation method of cough with asthma sheet of offer is provided.
It is an object of the invention to by following scheme realization:
Green and that cough with asthma sheet is in preparation suppression OS-732 cells MG-63 cell proliferation application, described green and cough with asthma Sheet by little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g makes as crude drug, and described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, chicken Vow rattan 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6% that entrainer accounts for the percent by volume of total extractant, extraction pressure Power 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min, obtain supercritical extraction Take thing, supercritical extract is added starch, 70% ethanol granule, it is dried, tabletting, makes 200, every tablet weight 0.50g.
Preferably, above-mentioned green and cough with asthma sheet is in preparation suppression OS-732 cells MG-63 cell proliferation Application, described green and cough with asthma sheet preparation method comprise the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2In supercritical extraction device, ethanol As entrainer, it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol system Granule, is dried, tabletting, makes 200, every tablet weight 0.50g.
In prior art, green and cough with asthma particulate oral, a 10g, 3-4 time on the one.Green and cough with asthma granule dosage is big.Adopt Green and every tablet weight 0.50g of cough with asthma sheet being prepared as by the inventive method, the most only needs 2, within 1st, takes 3-4 time.Have more Dose is greatly reduced under conditions of many active component.This conclusion can be proved by following test.
The comparison of emodin content in green and cough with asthma sheet prepared by test one, distinct methods
L, instrument and the reagent present invention be green and cough with asthma sheet: prepares by embodiment 1 method, uses 1000g crude drug, extracted makes 200, every tablet weight 0.50g.Former green and cough with asthma granule, according to WS-10616(ZD-0616) prepared by-2002 standard methods. Agilent1200 high performance liquid chromatograph；METTLER AE240 electronic analytical balance；(China's medicine is biological for rheum emodin reference substance Goods examine and determine institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia the 4th note on the use 15 of version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Methanol-0.1% phosphoric acid Solution (80: 20) is flowing phase；Detection wavelength is 437nm.Number of theoretical plate is calculated by rheum emodin peak should be not less than 4000.
It is appropriate that the preparation precision of reference substance solution weighs rheum emodin reference substance, adds methanol and makes molten containing 10 g of every 1ml Liquid, to obtain final product.
The preparation of need testing solution takes that the present invention is green and cough with asthma sheet 10, finely ground, and mixing takes 0.1g, accurately weighed, adds Water 20ml makes dissolving, adds hydrochloric acid 2ml, shakes up, and is heated to reflux 30 minutes, cooling, adds chloroform 30ml, supersound process 10 minutes, turns Entering in separatory funnel, divide and take chloroform solution, water liquid extracts 2 times with chloroform shaking again, and each 15ml, combined chloroform liquid, at 80 DEG C of water Being evaporated in bath, residue methanol dissolves and is transferred in 10ml measuring bottle, adds methanol dilution to scale, shakes up, use microporous filter membrane (0.45 m) filters, and takes filtrate, to obtain final product.
The preparation of reference product need testing solution takes the green of comparison and cough with asthma granule 20g, finely ground, mixing, takes 1g, accurate Weighed, the 20ml that adds water makes dissolving, adds hydrochloric acid 2ml, shakes up, and is heated to reflux 30 minutes, cooling, adds chloroform 30ml, supersound process 10 Minute, proceeding in separatory funnel, divide and take chloroform solution, water liquid extracts 2 times with chloroform shaking again, each 15ml, combined chloroform liquid, Being evaporated in 80 DEG C of water-baths, residue methanol dissolves and is transferred in 10ml measuring bottle, adds methanol dilution to scale, shakes up, use micropore Filter membrane (0.45 m) filters, and takes filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and need testing solution, each 20 μ l of reference product need testing solution, note Enter chromatograph of liquid, measure, to obtain final product.
3, result
Result shows, the present invention is green and in cough with asthma sheet, the content of rheum emodin is 1.65-3.28mg/ sheet；And former green and cough with asthma granule The content of middle rheum emodin is 1.62mg/ bag (every bag of 10g Han granule), and the emodin content each serving consumption 2 is former granule 2-4 times of agent 10g content, in the case of dose reduces, emodin content improves a lot.
The studies above shows, uses green and cough with asthma sheet prepared by preparation method of the present invention, and active constituent content is significantly larger than WS-10616(ZD-0616) green and cough with asthma granule prepared by the method that-2002 standards are recorded.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, in order to those skilled in the art knows more about The present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, all above-mentioned based on the present invention The technology that content is realized belongs to the scope of the present invention.
Embodiment 1
Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the volume of total extractant Percentage ratio is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, must surpass and face Boundary's extract, adds starch by supercritical extract, 70% ethanol granule, is dried, tabletting, makes 200, every tablet weight 0.50g。
After testing, in finished product, the content of rheum emodin is 3.28mg/ sheet.
Embodiment 2
Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the volume of total extractant Percentage ratio is 4%, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 900min, must surpass and face Boundary's extract, adds starch by supercritical extract, 70% ethanol granule, is dried, tabletting, makes 200, every tablet weight 0.50g。
After testing, in finished product, the content of rheum emodin is 1.65mg/ sheet.
Embodiment 3
Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the volume of total extractant Percentage ratio is 6%, extracting pressure 30MPa, temperature 60 C, CO2Flow 3ml/g crude drug min, extraction time 800min, must surpass and face Boundary's extract, adds starch by supercritical extract, 70% ethanol granule, is dried, tabletting, makes 200, every tablet weight 0.50g。
After testing, in finished product, the content of rheum emodin is 2.08mg/ sheet.
Embodiment 4: the green and experimentation data of cough with asthma sheet suppression people's OS-732 cells MG-63 cell proliferation
1. experiment material
1.1 experiment cell strains
People's OS-732 cells MG-63 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention is green and cough with asthma sheet: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh that 100mg is green and cough with asthma sheet, is dissolved in 5ml dehydrated alcohol, and 0.2 m filter filters, and 500 Ldoff pipe subpackage ,-20 DEG C of storages, 0.2 m filter filters dehydrated alcohol in case matched group is used simultaneously.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061 Lot.No.758137)；Hyclone (Hangzhoupro, sky, Zhejiang biotechnology Company limited Lot.No.100419)；NaHC03(the long hundred million chemical reagent company limited Cat.No.11810-033 in Shanghai Lot.No. 1088387)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)；Penicillin G Sodium Salt(AMRESCO company 1)；Streptomycin Sulfate(AMRESCO)；(Zibo Ya Dulan economy and trade is limited for dehydrated alcohol Company)；MTT (Biosharp lot number: 0793): PBS(laboratory autogamy)；
1.4 experiment equipment
Lycra inverted microscope (Germany Leica model: DMIL)；Visible-ultraviolet light microwell plate detector (MD company type of the U.S. Number: SPECTRAMAX 190)；C02Incubator (FORMA model: 3111)；Super-clean bench (safe and sound company of Su Jing group manufacture model: SW-CJ-ZFD)；Pure water instrument (Sprlng company of U.S. model: S/N 020579)；Accurate pipettor (Gilson Inc of France type Number: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；(Japan SANYO is public for full-automatic high-pressure autoclave Department model: MLS-3020)；Table electrothermal air dry oven (Shanghai precision 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 Model: KA-1000)；0.2 m filter (MILLIPORE model: SLGP033RB)；1cm culture dish (NEST company), 96 holes are cultivated Plate (NEST company)；Cell counting count board；Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) MG-63 cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when cell grows to During logarithmic (log) phase, collecting cell, discard culture fluid, PBS rinses 3 times, addition 3ml 0.25% trypsin-0.04%EDTA, 37 DEG C After digestion 2min, it is added thereto in 5ml complete medium and reacts, being proceeded in centrifuge tube after piping and druming cell, 1000rpm Centrifugal 5min, adjusts concentration of cell suspension 3 × 104Individual/ml.
2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 l, and culture plate is put in cell culture incubator (37 DEG C, 5%C02) cellar culture.
3) according to cell growth status, general long to 50%-70%, add green and cough with asthma sheet solution, continue to cultivate 24h.
4) add 20 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 adds 200 l dimethyl sulfoxide, puts shaking table Upper low-speed oscillation 10min, makes crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm.
6) arranging background (being not added with cell, only add culture fluid), (cell, the medicine dissolution of same concentrations are situated between control wells simultaneously Matter, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes.
7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD Value), control wells OD value × 100%.Experiment is repeated 3 times.
3. statistical disposition
Using the correlation analysis in Microsoft Excel 2007 software and Student t inspection, data are with mean ± S.D. Represent.
4. experimental result
Statistical result showed after mtt assay experiment, compares with matched group, when dosage reaches 5mg/ml, presses down MG-63 cell proliferation Being shaped with difference (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), when dosage reaches 15-20mg/ml Time have pole significant difference (P < 0.001).
Table 1 is green and cough with asthma sheet is to MG-63 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 12.17±4.58
2 10 24.12±6.56*
3 15 34.15±8.59**
4 20 40.49±11.68**
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
Green and the cough with asthma sheet of the present invention can suppress MG-63 cell proliferation, reduces the cell growing number of MG-63 cell, this work With in dose dependent.

Claims (2)

  1. The application in preparation suppression OS-732 cells MG-63 cell proliferation of the greenest and cough with asthma sheet, described green and cough Breathe heavily sheet by little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g make as crude drug, it is characterised in that described green and cough with asthma sheet preparation method comprises the steps: Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Huang Essence 100g, joins CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the percent by volume of total extractant For 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700- 800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, makes 200 Sheet, every tablet weight 0.50g.
  2. 2. according to the green and cough with asthma sheet described in claim l in preparation suppression OS-732 cells MG-63 cell proliferation In application, it is characterised in that described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO2Super In critical extractor, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, Temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, added by supercritical extract Enter starch, 70% ethanol granule, it is dried, tabletting, makes 200, every tablet weight 0.50g.
CN201610088722.6A 2016-02-17 2016-02-17 Application of pseudobulb-of-corymbose-coelogyne cough and asthma tablets to preparation of medicine for restraining human osteosarcoma cell MG-63 multiplication Withdrawn CN105726960A (en)

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凌汶静: "常用抗肿瘤中药的临床应用与规律探", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 *
国家药品监督管理局: "《国家中成药标准汇编内科肺系（二）分册》", 1 December 2002 *

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Application publication date: 20160706