CN105726650A - Application of Liuweishiliu tablet to preparation of drugs for inhibiting malignant brain glioma cell SHG-44 proliferation - Google Patents

Application of Liuweishiliu tablet to preparation of drugs for inhibiting malignant brain glioma cell SHG-44 proliferation Download PDF

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CN105726650A
CN105726650A CN201610190331.5A CN201610190331A CN105726650A CN 105726650 A CN105726650 A CN 105726650A CN 201610190331 A CN201610190331 A CN 201610190331A CN 105726650 A CN105726650 A CN 105726650A
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punica granatum
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sheet
cell
shg
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不公告发明人
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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)
    • 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)
    • 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)
    • A61K36/67—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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Abstract

The invention belongs to the technical field of traditional Chinese medicines and in particular relates to an application of a Liuweishiliu tablet to preparation of drugs for inhibiting malignant brain glioma cell SHG-44 proliferation and a preparation method of the Liuweishiliu tablet. The Liuweishiliu tablet is prepared by using 90g of pomegranate seed, 45g of cassia bark, 45g of netmeg, 45g of pepper, 45g of safflower and 45g of semen caesalpiniae cristae as raw medicines and is prepared by adopting supercritical extraction, so that the content of piperine is greatly increased.

Description

The application in preparation suppression Malignant cerebral gliomas cell SHG-44 cell proliferation of the Six-element Punica granatum L. sheet
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the application in preparation suppression Malignant cerebral gliomas cell SHG-44 cell proliferation of a kind of Six-element Punica granatum L. sheet 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 standard for traditional Chinese medicines compilation surgery gynecological of country fascicle.It is made up as crude drug of Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, there is the kidney warming, effect of dim waist knee joint.For women's leukorrhea.
In prior art, not yet there is the Six-element Punica granatum L. sheet report in the application in preparation suppression Human annexin Ⅴ cell SHG-44 cell proliferation, also there are no Six-element Punica granatum L. sheet and extract the report of preparation aspect employing supercritical extraction, and the method for conventional size reduction, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
Goal of the invention: it is an object of the invention to provide the application in preparation suppression Malignant cerebral gliomas cell SHG-44 cell proliferation of a kind of Six-element Punica granatum L. sheet.
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 Malignant cerebral gliomas cell SHG-44 cell proliferation of the Six-element Punica granatum L. sheet, described Six-element Punica granatum L. sheet is made up as crude drug of Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, 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, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6% that entrainer accounts for the percent by volume of total extractant, 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, is dried, tabletting, makes 500, every tablet weight 0.30g.
Preferably, the application in preparation suppression Malignant cerebral gliomas cell SHG-44 cell proliferation of the above-mentioned Six-element Punica granatum L. sheet, 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, joins CO2In supercritical extraction device, 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 350min, obtain supercritical extract, supercritical extract added starch, 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.Six-element every tablet weight 0.30g of Punica granatum L. sheet using the inventive method to be prepared as, the most only needs 2, within 1st, takes 3 times.Dose is greatly reduced under conditions of there is more active component.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；Piperine reference substance (Nat'l Pharmaceutical & Biological Products Control 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 makes 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, 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.
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, to obtain final product.
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 the content of piperine is 0.81mg/ grain in former Six-element Punica granatum L. capsule, 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) the Six-element Punica granatum L. capsule prepared of method recorded of-2002 standards.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that 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, and all technology realized based on foregoing of the present invention belong 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 CO2In supercritical extraction device, 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 350min, obtain supercritical extract, supercritical extract added starch, 70% ethanol glue capsule, 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 CO2In supercritical extraction device, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 300min, obtain supercritical extract, supercritical extract added starch, 70% ethanol glue capsule, 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 CO2In supercritical extraction device, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature 60 C, CO2Flow 3ml/g crude drug min, extraction time 400min, obtain supercritical extract, supercritical extract added starch, 70% ethanol glue capsule, 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 Human annexin Ⅴ cell SHG-44 cell proliferation
1. experiment material
1.1 experiment cell strains
Human annexin Ⅴ cell SHG-44 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, 500 ldoff pipe subpackages ,-20 DEG C of storages, and 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)；Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419)；NaHC03(the long hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 in Shanghai)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)；PenicillinGSodiumSalt(AMRESCO company 1)；StreptomycinSulfate(AMRESCO)；Dehydrated alcohol (Zibo Ya Dulan 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 of U.S. model: 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 (Gilson Inc of France model: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；Full-automatic high-pressure autoclave (SANYO company of Japan 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's model: KA-1000)；0.2 m 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) SHG-44 cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when cell grows to logarithmic (log) phase, collecting cell, discard culture fluid, PBS rinses 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, it is added thereto in 5ml complete medium and reacts, being proceeded in centrifuge tube after piping and druming cell, 1000rpm is centrifuged 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, culture plate 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 low-speed oscillation 10min on shaking table, 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), control wells (cell, the medicine dissolution medium of same concentrations, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes simultaneously.
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 represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, variant to SHG-44 cell inhibitory effect (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 Six-element Punica granatum L. sheet is to SHG-44 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 10.14±3.02
2 10 19.32±6.59*
3 15 37.26±9.02**
4 20 45.44±11.29**
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 SHG-44 cell proliferation, reduces the cell growing number of SHG-44 cell, and this effect is dose dependent.

Claims (2)

1. Six-element Punica granatum L. sheet application in preparation suppression Malignant cerebral gliomas cell SHG-44 cell proliferation, described Six-element Punica granatum L. sheet is made up as crude drug of Semen Granati 90g, Cortex Cinnamomi 45g, Semen Myristicae 45g, Fructus Piperis 45g, Flos Carthami 45g, Ramulus seu Fructus Caesalpiniae 45g, it is characterized 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, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6% that entrainer accounts for the percent by volume of total extractant, 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, is dried, tabletting, makes 500, every tablet weight 0.30g.
2. preparing, according to the Six-element Punica granatum L. sheet described in claim l, the application suppressed in Malignant cerebral gliomas cell SHG-44 cell proliferation, it is characterized 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, joins CO2In supercritical extraction device, 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 350min, obtain supercritical extract, supercritical extract added starch, 70% ethanol glue capsule, is dried, tabletting, makes 500, every tablet weight 0.30g.
CN201610190331.5A 2016-03-30 2016-03-30 Application of Liuweishiliu tablet to preparation of drugs for inhibiting malignant brain glioma cell SHG-44 proliferation Withdrawn CN105726650A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621632A (en) * 2021-08-06 2021-11-09 河南大学 Method for knocking down CBS gene and application of method in preparation of medicine for treating human brain glioma

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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621632A (en) * 2021-08-06 2021-11-09 河南大学 Method for knocking down CBS gene and application of method in preparation of medicine for treating human brain glioma
CN113621632B (en) * 2021-08-06 2023-06-06 河南大学 Method for knocking down CBS gene and application of method in preparation of medicine for treating human brain glioma

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