CN105832825A - Use of tablet of six ingredients with pomegranate seeds in preparation of drug for inhibiting brain tumor cell SF126 proliferation - Google Patents

Use of tablet of six ingredients with pomegranate seeds in preparation of drug for inhibiting brain tumor cell SF126 proliferation Download PDF

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Publication number
CN105832825A
CN105832825A CN201610190346.1A CN201610190346A CN105832825A CN 105832825 A CN105832825 A CN 105832825A CN 201610190346 A CN201610190346 A CN 201610190346A CN 105832825 A CN105832825 A CN 105832825A
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punica granatum
preparation
sheet
ethanol
semen
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CN201610190346.1A
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杨志华
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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Jinan Xinshidai Medicine Science and 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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
    • A61K36/286—Carthamus (distaff thistle)
    • 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
    • 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)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • 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)
    • A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
    • 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
    • A61K2236/37—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
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Alternative & Traditional Medicine (AREA)
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  • Microbiology (AREA)
  • Medical Informatics (AREA)
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  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention belongs to the technical field of traditional Chinese medicines and relates to a use and a preparation method of a tablet of six ingredients with pomegranate seeds in preparation of a drug for inhibiting brain tumor cell SF126 proliferation. The tablet of six ingredients with pomegranate seeds is prepared from 90g of pomegranate seeds, 45g of cassia bark, 45g of semen myristicae, 45g of pepper, 45g of safflower and 45g of semen caesalpiniae cristae. A supercritical extraction method greatly improves piperine content.

Description

Six-element Punica granatum L. sheet is in preparation suppression Tumor cells SF126 cell proliferation Application
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Six-element Punica granatum L. sheet at preparation suppression Tumor cells SF126 Application in cell proliferation and the preparation method of Six-element Punica granatum L. sheet.
Background technology
Six-element Punica granatum L. capsule standard WS-10474(ZD-0474)-2002, it is recorded in country's standard for traditional Chinese medicines compilation surgery Gynecological's fascicle.By Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g as raw material Medicine is made, and has the kidney warming, effect of dim waist knee joint.For women's leukorrhea.
In prior art, not yet there is Six-element Punica granatum L. sheet in preparation suppression SHG44 cells cell SF126 cell proliferation The report of application, also there are no Six-element Punica granatum L. sheet and extract preparation aspect and use the report of supercritical extraction, and conventional size reduction Method, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, it has not been convenient to take, has had a strong impact on this product clinically Application.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of Six-element Punica granatum L. sheet thin at preparation suppression Tumor cells SF126 Application in born of the same parents' hyperproliferation agent.
Further object is that the preparation method that a kind of Six-element Punica granatum L. sheet is provided.
It is an object of the invention to by following scheme realization:
The application in preparation suppression Tumor cells SF126 cell proliferation of the Six-element Punica granatum L. sheet, described Six-element Punica granatum L. sheet is by stone Pomegranate 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g make as crude drug, and described six The preparation method of taste Punica granatum L. sheet comprise the steps: to take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, joins CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the volume of total extractant Percentage ratio is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 300-400min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol glue capsule, is dried, tabletting, makes 500, every tablet weight 0.30g.
Preferably, the application in preparation suppression Tumor cells SF126 cell proliferation of the above-mentioned Six-element Punica granatum L. sheet, institute The preparation method stating Six-element Punica granatum L. sheet comprises the steps: to take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extractant Percent by volume is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 350min, Supercritical extract, adds starch by supercritical extract, 70% ethanol glue capsule, is dried, tabletting, makes 500, every tablet weight 0.30g。
In prior art, Six-element Punica granatum L. capsule oral, one time 5,3 times on the one.Six-element Punica granatum L. capsule dosage is big.Use Six-element every tablet weight 0.30g of Punica granatum L. sheet that the inventive method is prepared as, the most only needs 1, within 1st, takes 3 times.There is more work Dose is greatly reduced under conditions of property composition.This conclusion can be proved by following test.
The comparison of content of piperine in Six-element Punica granatum L. sheet prepared by test one, distinct methods
L, instrument and reagent Six-element of the present invention Punica granatum L. sheet: prepare by embodiment 1 method, uses 315g crude drug, extracted makes 500, every tablet weight 0.30g.Former Six-element Punica granatum L. capsule, according to WS-10474(ZD-0474) prepared by-2002 standard methods. Agilent1200 high performance liquid chromatograph；METTLERAE240 electronic analytical balance；(China's medicine is biological for piperine 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；With methanol-water (77: 23) for flowing phase；Detection wavelength is 343nm.Number of theoretical plate is calculated by piperine peak should be not less than 1500.
It is appropriate that the preparation precision of reference substance solution weighs piperine reference substance, puts in brown measuring bottle, adds dehydrated alcohol and make Every 1ml solution containing 20 g, to obtain final product.
The preparation of the need testing solution of the Six-element Punica granatum L. sheet of the present invention takes the Six-element Punica granatum L. sheet 6g of the present invention, mixing, takes 0.12g, accurately weighed, put in 50ml brown measuring bottle, add dehydrated alcohol 40ml, supersound process 30 minutes, let cool, add dehydrated alcohol It is diluted to scale, shakes up, filter, take subsequent filtrate, to obtain final product.
The preparation of reference product need testing solution takes comparison Six-element Punica granatum L. capsule 6g, mixing, takes 0.3g, accurately weighed, puts In 50ml brown measuring bottle, add dehydrated alcohol 40ml, supersound process 30 minutes, let cool, add dehydrated alcohol and be diluted to scale, shake up, Filter, take subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 10 l of need testing solution, injects chromatograph of liquid, measures, Obtain.
3, result
Result shows, in Six-element Punica granatum L. sheet of the present invention, the content of piperine is 4.10-8.05mg/ sheet；And former Six-element Punica granatum L. capsule The content of middle piperine is 0.81mg/ grain, each serving 2-4 times that content of piperine is 5 content of original capsule of consumption 2, In the case of dose reduces, content of piperine improves a lot.
The studies above shows, uses Six-element Punica granatum L. sheet prepared by preparation method of the present invention, and active constituent content is significantly larger than WS-10474(ZD-0474) Six-element Punica granatum L. capsule 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 Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2Surpass and face In boundary's extractor, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature Spend 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 350min, obtain supercritical extract, is added by supercritical extract Starch, 70% ethanol glue capsule, it is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of piperine is 8.05mg/ sheet.
Embodiment 2
Take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2Surpass and face In boundary's extractor, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature Spend 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 300min, obtain supercritical extract, is added by supercritical extract Starch, 70% ethanol glue capsule, it is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of piperine is 4.15mg/ sheet.
Embodiment 3
Take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2Surpass and face In boundary's extractor, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature Spend 60 DEG C, CO2Flow 3ml/g crude drug min, extraction time 400min, obtain supercritical extract, is added by supercritical extract Starch, 70% ethanol glue capsule, it is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of piperine is 6.03mg/ sheet.
Embodiment 4: the experimentation data of Six-element Punica granatum L. sheet suppression SHG44 cells cell SF126 cell proliferation
1. experiment material
1.1 experiment cell strains
SHG44 cells cell SF126 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Six-element Punica granatum L. sheet of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh 100mg Six-element Punica granatum L. sheet, be dissolved in 5ml dehydrated alcohol, 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.12100-061Lot.No.758137)；(Hangzhoupro, sky, Zhejiang biotechnology has hyclone Limit company Lot.No.100419)；NaHC03(the long hundred million chemical reagent company limited Cat.No.11810-in Shanghai 033Lot.No.1088387)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)； PenicillinGSodiumSalt(AMRESCO company 1)；StreptomycinSulfate(AMRESCO)；Dehydrated alcohol is (black Bo Yadulan Trade Co., Ltd.)；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: SPECTRAMAX190)；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/N020579)；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) SF126 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, adds 3ml0.25% trypsin-0.04%EDTA, and 37 DEG C disappear Change after 2min, be added thereto in 5ml complete medium and react, proceeded in centrifuge tube after blowing and beating cell, 1000rpm from Heart 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 Six-element Punica granatum L. sheet solution, continue to cultivate 24h.
4) add 20 lMTT 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 MicrosoftExcel2007 software and Studentt inspection, data are with mean ± S.D. table Show.
4. experimental result
Statistical result showed after mtt assay experiment, compares with matched group, when dosage reaches 5mg/ml, presses down SF126 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 Six-element Punica granatum L. sheet is to SF126 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 10.53±2.96
2 10 18.98±4.59*
3 15 26.33±7.06**
4 20 34.28±10.02**
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
The Six-element Punica granatum L. sheet of the present invention can suppress SF126 cell proliferation, reduces the cell growing number of SF126 cell, this work With in dose dependent.

Claims (2)

1. Six-element Punica granatum L. sheet preparation suppression Tumor cells SF126 cell proliferation in application, described Six-element Punica granatum L. sheet by Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g make as crude drug, and it is special Levying and be, the preparation method of described Six-element Punica granatum L. sheet comprises the steps: to take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, recklessly Green pepper 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2In supercritical extraction device, ethanol accounts for always as entrainer, entrainer The percent by volume of extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug Min, extraction time 300-400min, obtain supercritical extract, supercritical extract added starch, 70% ethanol glue capsule, dry Dry, tabletting, make 500, every tablet weight 0.30g.
2. according to the answering in preparation suppression Tumor cells SF126 cell proliferation of the Six-element Punica granatum L. sheet described in claim l With, it is characterised in that the preparation method of described Six-element Punica granatum L. sheet comprises the steps: to take Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, join CO2In supercritical extraction device, ethanol, as entrainer, is carried secretly It is 5% that agent accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, Extraction time 350min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol glue capsule, is dried, tabletting, Make 500, every tablet weight 0.30g.
CN201610190346.1A 2016-03-30 2016-03-30 Use of tablet of six ingredients with pomegranate seeds in preparation of drug for inhibiting brain tumor cell SF126 proliferation Withdrawn CN105832825A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1840133A (en) * 2006-01-21 2006-10-04 端智 Six-ingredient tablet containing pomegranate
CN103566023A (en) * 2013-10-29 2014-02-12 山东省立医院 Preparation method and application of toothache anti-inflammation tablets

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1840133A (en) * 2006-01-21 2006-10-04 端智 Six-ingredient tablet containing pomegranate
CN103566023A (en) * 2013-10-29 2014-02-12 山东省立医院 Preparation method and application of toothache anti-inflammation tablets

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