CN105641449A - Application of antitussive and antiasthmatic tablets containing bulbophyllum in preparation of drugs for inhibiting proliferation of melanoma cells MM96L - Google Patents
Application of antitussive and antiasthmatic tablets containing bulbophyllum in preparation of drugs for inhibiting proliferation of melanoma cells MM96L Download PDFInfo
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- 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)
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- 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)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
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- A61K36/704—Polygonum, e.g. knotweed
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- A61K36/18—Magnoliophyta (angiosperms)
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- 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
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- 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
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Abstract
The invention belongs to the technical field of traditional Chinese medicines and particularly relates to an application of antitussive and antiasthmatic tablets containing bulbophyllum in preparation of drugs for inhibiting proliferation of melanoma cells MM96L as well as a preparation method of the antitussive and antiasthmatic tablets containing bulbophyllum. The antitussive and antiasthmatic tablets containing bulbophyllum are prepared from raw material medicines including 200 g of bulbophyllum, 200 g of Chinese fevervine herbs, 100 g of leatherleaf mahonia stems, 100 g of marsdenia tenacissima, 100 g of common bletilla pseudobulb, 100 g of giant knotweed rhizomes, 100 g of garden balsam stems and 100 g of manyflower solomonseal rhizomes. The antitussive and antiasthmatic tablets containing bulbophyllum are prepared through supercritical extraction, so that the content of rheum emodin is greatly increased.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, be specifically related to a kind of green and cough and breathe heavily sheet and preparing check melanin oncocyteApplication in MM96L cell proliferation medicine and green and cough the preparation method who breathes heavily sheet.
Background technology
Green and cough and breathe heavily particle standard No. WS-10616(ZD-0616)-2002, be recorded in national standard for traditional Chinese medicines compilation internal medicineLung system (two) fascicle. By little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g,Lopseed 100g, sealwort 100g make as bulk drug, have effect of Yin nourishing and lung moistening, clearing heat and detoxicating, removing blood stasis and hemostasis, forThe cough that the dry criminal's lung of heat causes, hectic fever, the diseases such as night sweat.
In prior art, not yet have green and cough and breathe heavily sheet and suppressing human melanoma cell MM96L cell proliferation medicine in preparationThe report of the application in thing, also there are no green and cough and breathe heavily sheet and extract preparation aspect and adopt the report of supercritical extract, and traditional waterThe method of decoct, alcohol reflux extracting, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, is inconvenient to take, seriousHaving affected this product applies clinically.
Summary of the invention
Goal of the invention: the object of the present invention is to provide a kind of green and cough and breathe heavily sheet and preparing check melanin oncocyteApplication in MM96L cell proliferation medicine.
It is a kind of green and cough the preparation method who breathes heavily sheet that another object of the present invention is to provide.
The object of the invention is to realize by following scheme:
Green and cough and breathe heavily sheet in the application of preparing in check melanin oncocyte MM96L cell proliferation medicine, described green and cough and breathe heavily sheetBy little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed 100g, HuangEssence 100g makes as bulk drug, described green and cough that the preparation method who breathes heavily sheet comprises the steps: to get little green splendid achnatherum 200g, chicken is vowedRattan 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed 100g, sealwort 100g, join CO2In supercritical extract device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min, obtains supercritical extractThing, adds starch by supercritical extract, 70% ethanol particle processed, and dry, compressing tablet, makes 200, every heavy 0.50g.
Preferably, above-mentioned green and cough breathe heavily sheet prepare in check melanin oncocyte MM96L cell proliferation medicine shouldWith, described green and cough the preparation method who breathes heavily sheet and comprise the steps: to get little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, logicalClose rattan 100g, bletilla 100g, giant knotweed 100g, lopseed 100g, sealwort 100g, join CO2In supercritical extract device, ethanol is doneFor entrainer, the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa, 40 DEG C of temperature, CO2Flow2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is added to starch, 70% ethanol systemParticle, dry, compressing tablet, makes 200, every heavy 0.50g.
In prior art, green and cough and breathe heavily particulate oral, a 10g, 3-4 time on the one. Green and cough that to breathe heavily particle dosage large. AdoptBe prepared into by the inventive method green and cough and breathe heavily every heavy 0.50g of sheet only needs 2 at every turn, within 1st, takes 3-4 time. Have moreUnder the condition of many active components, greatly reduce dose. This conclusion can be by following evidence.
Test one, prepare green of distinct methods and cough the comparison of breathing heavily emodin content in sheet
L, instrument and reagent the present invention are green and cough and breathe heavily sheet: by embodiment 1 method preparation, use 1000g bulk drug, make through extracting200, every heavy 0.50g. Former green and cough and breathe heavily particle, according to WS-10616(ZD-0616)-2002 standard methods preparations.Agilent1200 high performance liquid chromatograph; METTLERAE240 electronic analytical balance; Archen reference substance (Chinese medicine biologyGoods calibrating institute).
2, method
Measure according to high performance liquid chromatography (the 4th note on the use 15 of Chinese pharmacopoeia version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Methyl alcohol-0.1% phosphorusAcid solution (80: 20) is mobile phase; Detection wavelength is 437nm. Number of theoretical plate calculates and should be not less than 4000 by archen peak.
It is appropriate that the preparation precision of reference substance solution takes archen reference substance, adds methyl alcohol and make molten containing 10 μ g of every 1mlLiquid, to obtain final product.
It is green and cough and breathe heavily 10 of sheets that the present invention is got in the preparation of need testing solution, and porphyrize, mixes, and gets 0.1g, accurately weighed, addsWater 20ml makes to dissolve, and adds hydrochloric acid 2ml, shakes up, and adds hot reflux 30 minutes, cooling, adds chloroform 30ml, and ultrasonic processing 10 minutes, turnsEnter in separatory funnel, divide and get chloroform solution, water liquid extracts 2 times with chloroform jolting again, each 15ml, and combined chloroform liquid, at 80 DEG C of waterEvaporate to dryness in bath, residue dissolves with methyl alcohol and is transferred in 10ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and uses miillpore filter(0.45 μ m) filters, and gets filtrate, to obtain final product.
The preparation of reference product need testing solution is got the green of contrast and is coughed and breathe heavily particle 20g, and porphyrize, mixes, and gets 1g, precisionWeighed, the 20ml that adds water makes to dissolve, and adds hydrochloric acid 2ml, shakes up, and adds hot reflux 30 minutes, cooling, adds chloroform 30ml, ultrasonic processing 10Minute, proceed in separatory funnel, divide and get chloroform solution, water liquid extracts 2 times with chloroform jolting again, each 15ml, combined chloroform liquid,Evaporate to dryness in 80 DEG C of water-baths, residue dissolves with methyl alcohol and is transferred in 10ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, and uses micropore(0.45 μ m) filters filter membrane, gets filtrate, to obtain final product.
Determination method difference accurate absorption reference substance solution and need testing solution, the each 20 μ l of reference product need testing solution,Injection liquid chromatography, measures, and to obtain final product.
3, result
Result shows, the present invention is green and to cough the content of breathing heavily archen in sheet be 1.65-3.28mg/ sheet; And former green and cough and breathe heavily particleThe content of middle archen is 1.62mg/ bag (every bag containing granule 10g), is former particle each serving the emodin content of 2 of consumptionsDoubly, in the situation that dose reduces, emodin content improves a lot the 2-4 of agent 10g content.
Above-mentioned research shows, adopt prepared by preparation method of the present invention green and cough and breathe heavily sheet, active constituent content far away higher thanWS-10616(ZD-0616) prepare green of method that-2002 standards are recorded and cough and breathe heavily particle.
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 understandsThe present invention, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example, all above-mentioned based on the present inventionThe technology that content realizes all belongs to scope of the present invention.
Embodiment 1
Get little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed100g, sealwort 100g, join CO2In supercritical extract device, ethanol is as entrainer, and entrainer accounts for the volume of total extractantPercentage is 5%, extracting pressure 25MPa, 40 DEG C of temperature, CO2Flow 2ml/g crude drug min, extraction time 750min, must surpass and faceBoundary's extract, adds starch by supercritical extract, 70% ethanol particle processed, and dry, compressing tablet, makes 200, every weight0.50g。
After testing, in finished product, the content of archen is 3.28mg/ sheet.
Embodiment 2
Get little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed100g, sealwort 100g, join CO2In supercritical extract device, ethanol is as entrainer, and entrainer accounts for the volume of total extractantPercentage is 4%, extracting pressure 15MPa, 30 DEG C of temperature, CO2Flow 1ml/g crude drug min, extraction time 900min, must surpass and faceBoundary's extract, adds starch by supercritical extract, 70% ethanol particle processed, and dry, compressing tablet, makes 200, every weight0.50g。
After testing, in finished product, the content of archen is 1.65mg/ sheet.
Embodiment 3
Get little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed100g, sealwort 100g, join CO2In supercritical extract device, ethanol is as entrainer, and entrainer accounts for the volume of total extractantPercentage is 6%, extracting pressure 30MPa, temperature 60 C, CO2Flow 3ml/g crude drug min, extraction time 800min, must surpass and faceBoundary's extract, adds starch by supercritical extract, 70% ethanol particle processed, and dry, compressing tablet, makes 200, every weight0.50g。
After testing, in finished product, the content of archen is 2.08mg/ sheet.
Embodiment 4: green and cough and breathe heavily the experimental study data that sheet suppresses human melanoma cell MM96L cell proliferation
1. experiment material
1.1 experiment cell lines
Human melanoma cell MM96L cell, Shandong University's laboratory cell bank, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention is green and cough and breathe heavily sheet: by embodiment 1 method preparation.
Liquid liquid storage: take 100mg green and cough and breathe heavily sheet, be dissolved in 5ml absolute ethyl alcohol, 0.2 μ m filter filters, 500 μThe packing of ldoff pipe ,-20 DEG C of storages, 0.2 μ m filter filters the use of absolute ethyl alcohol in order to control group simultaneously.
1.3 experiment reagent
DMEM (Cat.No.12100-061Lot.No.758137 of GIBCO company); Hyclone (Hangzhoupro, sky, Zhejiang biotechnologyThe Lot.No.100419 of Co., Ltd); NaHC03(Shanghai hundred million Cat.No.11810-033 of chemical reagent Co., Ltd of a specified durationLot.No.1088387); Trypsin(AMRESCO company); EDTA(AMRESCO company); PenicillinGSodiumSalt(AMRESCO company 1); StreptomycinSulfate (AMRESCO); (Zibo Ya Dulan economy and trade is limited for absolute ethyl alcoholCompany); 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 type of the U.S.Number: SPECTRAMAX190); C02Incubator (FORMA model: 3111); Super-clean bench (safe and sound company of Su Jing group manufactures model:SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. model: S/N020579); Accurate pipettor (French Gilson Inc typeNumber: P2); Electronic balance (German Sai Duolisi Co., Ltd model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO public affairsModel: the MLS-3020 of department); 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, ShanghaiModel: KA-1000); 0.2 μ m filter (MILLIPORE model: SLGP033RB); Cultivate in 1cm culture dish (NEST company), 96 holesPlate (NEST company); Cell counting count board; Centrifuge tube, pipette, Tips are some.
2. experimental technique
1) MM96L cell uses DMEM+10%FBS in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when Growth of Cells extremelyWhen logarithmic phase, collecting cell, discards nutrient solution, and PBS fine laundering 3 times, adds 3ml0.25% trypsase-0.04%EDTA, 37 DEG CAfter digestion 2min, add wherein 5ml complete medium neutralization reaction, after piping and druming cell, proceeded in centrifuge tube 1000rpmCentrifugal 5min, adjusts concentration of cell suspension 3 × 104Individual/ml.
2) cell kind is entered in 96 well culture plates, every hole adds cell suspension 180 μ l, and culture plate is put into cell culture incubator(37℃,5%C02) cellar culture.
3) according to Growth of Cells situation, generally grow to 50%-70%, add green and cough and breathe heavily sheet solution, continue to cultivate 24h.
4) after 24h, add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT), continue to cultivate 4h.
5) after 4h, buckle method is removed supernatant, pats dry gently with blotting paper, and every hole adds 200 μ l dimethyl sulfoxide (DMSO)s, puts shaking tableUpper low-speed oscillation 10min, fully dissolves crystal. Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add nutrient solution) is set simultaneously, (medicine dissolving of cell, same concentrations is situated between control wellsMatter, nutrient solution, MTT, dimethyl sulfoxide (DMSO)), set 6 multiple holes for every group.
7) result represents the inhibiting rate of cell with medicine: cell increment inhibiting rate (%)=(control wells OD value-dosing holes ODValue), control wells OD value × 100%. Experiment repeats 3 times.
3. statistical disposition
Adopt correlation analysis and Studentt inspection in MicrosoftExcel2007 software, data are with mean ± S.D.Represent.
4. experimental result
Statistical result showed after mtt assay experiment, with control group comparison, in the time that dosage reaches 5mg/ml, presses down MM96L cell proliferationBe shaped with difference (P < 0.05), dosage this difference in the time of 10mg/ml has conspicuousness (P < 0.01), when dosage reaches 15-20mg/mlTime have a utmost point significant difference (P < 0.001).
Table 1 is green and cough and breathe heavily sheet to MM96L cell inhibitory effect impact research (X ± SD)
Group | Drug concentration (mg/ml) | Inhibiting rate (%) |
Control group | 0 | 0 |
1 | 5 | 12.06±4.65 |
2 | 10 | 24.02±6.86* |
3 | 15 | 35.26±8.06** |
4 | 20 | 44.06±11.06** |
Note: with control group comparison, * P < 0.01; * P < 0.001.
5. experiment conclusion
Of the present invention green and cough and breathe heavily sheet and can suppress MM96L cell proliferation, reduce the Growth of Cells number of MM96L cell, this workWith being dose dependent.
Claims (2)
1. green and cough and breathe heavily sheet in the application of preparing in check melanin oncocyte MM96L cell proliferation medicine, described green and cough and breathe heavilySheet by little green splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed 100g,Sealwort 100g makes as bulk drug, it is characterized in that, described green and cough the preparation method who breathes heavily sheet and comprise the steps: to get littleGreen splendid achnatherum 200g, fevervine 200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed 100g, sealwort100g, joins CO2In supercritical extract device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min,Obtain supercritical extract, supercritical extract is added to starch, 70% ethanol particle processed, dry, compressing tablet, makes 200, everyHeavy 0.50g.
2. according to green described in claim l and cough and breathe heavily sheet preparing in check melanin oncocyte MM96L cell proliferation medicineApplication, it is characterized in that, described green and cough the preparation method who breathes heavily sheet and comprise the steps: to get little green splendid achnatherum 200g, fevervine200g, leatherleaf mahonia 100g, CAULIS MARSDENIAE TENACISSIMAE 100g, bletilla 100g, giant knotweed 100g, lopseed 100g, sealwort 100g, join CO2SuperIn critical extractor, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa,40 DEG C of temperature, CO2Flow 2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is addedEnter starch, 70% ethanol particle processed, dry, compressing tablet, makes 200, every heavy 0.50g.
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CN103494878A (en) * | 2013-10-08 | 2014-01-08 | 南京正亮医药科技有限公司 | Preparation method and application of compound danshen tablets |
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CN103494878A (en) * | 2013-10-08 | 2014-01-08 | 南京正亮医药科技有限公司 | Preparation method and application of compound danshen tablets |
Non-Patent Citations (2)
Title |
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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: 20160608 |