CN105616562A - Application of Tianhusui Yugan tablet to preparation of cell proliferation drug for restraining prostate cancer cells Du-145 - Google Patents

Application of Tianhusui Yugan tablet to preparation of cell proliferation drug for restraining prostate cancer cells Du-145 Download PDF

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Publication number
CN105616562A
CN105616562A CN201610183033.3A CN201610183033A CN105616562A CN 105616562 A CN105616562 A CN 105616562A CN 201610183033 A CN201610183033 A CN 201610183033A CN 105616562 A CN105616562 A CN 105616562A
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preparation
tianhusui yugan
yugan tablets
prostate cancer
cell proliferation
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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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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)
    • A61K36/23—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)
    • 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)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • 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

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
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  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
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  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
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  • Medicines Containing Plant Substances (AREA)

Abstract

The invention belongs to the technical field of traditional Chinese medicines and particularly relates to application of a Tianhusui Yugan tablet to the preparation of a cell proliferation drug for restraining prostate cancer cells Du-145 and a preparation method of the Tianhusui Yugan tabletl. The Tianhusui Yugan tablet is made of 699 g of hydrocotyle sibthorpioides, 1398 g of pimpinella candolleana, 699 g of oxalis corniculata and 209 g of anemone rivularis as raw material medicines and is prepared by supercritical extraction, so that the content of oleanolic acid is greatly improved.

Description

Tianhusui Yugan tablets suppresses the application in prostate cancer cell Du-145 cell proliferation in preparation
Technical field
The invention belongs to technical field of traditional Chinese medicines, it is specifically related to the preparation method that a kind of Tianhusui Yugan tablets suppresses the application in prostate cancer cell Du-145 cell proliferation and Tianhusui Yugan tablets in preparation.
Background technology
Tianhusui Yugan tablets standard No. WS-10213(ZD-0213)-2002, it is recorded in country's Chinese patent medicine standard compilation internal medicine liver and gall fascicle. It is made up as bulk drug of Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, has clearing heat and detoxicating, effect of depressed liver-energy dispersing and function of gallbladder promoting, for the acute and chronic hepatitis caused by dampness-heat in the liver and gallbladder.
In prior art, not yet there is Tianhusui Yugan tablets at the report suppressing the application in Human Prostate Cancer Cells Du-145 cell proliferation in preparation, also extract, there are no Tianhusui Yugan tablets, the report that preparation aspect adopts supercritical extraction, and the method that traditional water decoction is boiled, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, seriously have impact on this product and apply clinically.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of Tianhusui Yugan tablets and suppress the application in prostate cancer cell Du-145 cell proliferation in preparation.
Another object of the present invention is to provide the preparation method of a kind of Tianhusui Yugan tablets.
It is an object of the invention to by following scheme realize:
Tianhusui Yugan tablets suppresses the application in prostate cancer cell Du-145 cell proliferation in preparation, described Tianhusui Yugan tablets is made up as bulk drug of Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, the preparation method of described Tianhusui Yugan tablets comprises the steps: to get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, joins CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 4-6%, extracting pressure 15-30MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 800-1000min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
Preferably, above-mentioned Tianhusui Yugan tablets suppresses the application in prostate cancer cell Du-145 cell proliferation in preparation, the preparation method of described Tianhusui Yugan tablets comprises the steps: to get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, joins CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 5%, extracting pressure 25MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 900min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
In prior art, Tianhusui Yugan tablets is oral, one time 6,3 times on the one. Tianhusui Yugan tablets dosage is big. The heavy 0.30g of the every sheet of the Tianhusui Yugan tablets adopting the inventive method to be prepared into, only needs 3 every time, within 1st, takes 3 times. Dose is greatly reduced when having more activeconstituentss. This conclusion can be proved by following test.
The comparison of Oleanolic Acid content in Tianhusui Yugan tablets prepared by test one, different methods
L, instrument and reagent Tianhusui Yugan tablets of the present invention: prepare by embodiment 1 method, it may also be useful to 3005g bulk drug, makes 500 through extracting, the heavy 0.30g of every sheet. Former Tianhusui Yugan tablets, according to WS-10213(ZD-0213)-2002 standard method preparations. Agilent1200 high performance liquid chromatograph; METTLERAE240 electronic analytical balance; Oleanolic Acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2015 the 4th note on the use 15).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are weighting agent; Acetonitrile-0.1% phosphoric acid (80:20) is moving phase; Determined wavelength is 210nm. Theoretical stage number calculates by Oleanolic Acid peak should be not less than 3000.
It is appropriate that the preparation precision of reference substance solution takes Oleanolic Acid reference substance, adds methyl alcohol and makes the solution of every 1ml containing 0.2mg, to obtain final product.
The Tianhusui Yugan tablets of the present invention is got in the preparation of the Tianhusui Yugan tablets need testing solution of the present invention, grind thin, get the amount of 0.75g(2.5 sheet), accurately weighed, put in tool plug Erlenmeyer flask, precision adds ethanol 25ml, close plug, weighed weight, reflux 1 hour, let cool, weighed weight again, the weight of less loss is supplied with ethanol, shake even, filter, precision measures continuous filtrate 10ml, the hydrochloric acid soln 5ml that adds 20%, reflux 2.5 hours, add water 30ml, water-bath concentrates to without alcohol taste, let cool, 3 (40ml are extracted with methylene dichloride jolting, 30ml, 30ml), combined dichloromethane extracting solution, steam dry, residue adds methyl alcohol makes dissolving in right amount, it is transferred in 10ml measuring bottle, add methyl alcohol and it is diluted to scale, shake even, obtain.
The Tianhusui Yugan tablets 20g of comparison is got in the preparation of reference product need testing solution, grind thin, get the amount of 1.5g(5 sheet), accurately weighed, put in tool plug Erlenmeyer flask, precision adds ethanol 25ml, close plug, weighed weight, reflux 1 hour, let cool, weighed weight again, the weight of less loss is supplied with ethanol, shake even, filter, precision measures continuous filtrate 10ml, the hydrochloric acid soln 5ml that adds 20%, reflux 2.5 hours, add water 30ml, water-bath concentrates to without alcohol taste, let cool, 3 (40ml are extracted with methylene dichloride jolting, 30ml, 30ml), combined dichloromethane extracting solution, steam dry, residue adds methyl alcohol makes dissolving in right amount, it is transferred in 10ml measuring bottle, add methyl alcohol and it is diluted to scale, shake even, obtain.
Assay method is accurate Tianhusui Yugan tablets need testing solution, each 10 �� l of reference product need testing solution drawing reference substance solution and the present invention respectively, and injection liquid chromatography, measures, to obtain final product.
3, result
Result shows, in Tianhusui Yugan tablets of the present invention, the content of Oleanolic Acid is 3.22-6.45mg/ sheet; And the content of Oleanolic Acid is 0.81mg/ sheet in former Tianhusui Yugan tablets, each serving the Oleanolic Acid content of consumption 3 be former tablet 6 content 2-4 doubly, when dose reduces, Oleanolic Acid content improves a lot.
Above-mentioned research shows, adopts Tianhusui Yugan tablets prepared by preparation method of the present invention, and active constituent content is far away higher than WS-10213(ZD-0213) Tianhusui Yugan tablets prepared of method recorded of-2002 standards.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described, so that the technician of this area more understands 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 following example, and all technology realized based on foregoing of the present invention all belong to the scope of the present invention.
Embodiment 1
Get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, join CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 5%, extracting pressure 25MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 900min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
After testing, in finished product, the content of Oleanolic Acid is 6.45mg/ sheet.
Embodiment 2
Get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, join CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 4%, extracting pressure 15MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 1000min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
After testing, in finished product, the content of Oleanolic Acid is 4.98mg/ sheet.
Embodiment 3
Get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, join CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 6%, extracting pressure 30MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 60 DEG C, CO2Flow 3ml/g crude drug min, extraction time 800min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
After testing, in finished product, the content of Oleanolic Acid is 3.22mg/ sheet.
Embodiment 4: Tianhusui Yugan tablets suppresses the experiment research data of Human Prostate Cancer Cells Du-145 cell proliferation
1. experiment material
1.1 experiment cell strain
Human Prostate Cancer Cells Du-145 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Research medicine: Tianhusui Yugan tablets of the present invention: prepare by embodiment 1 method.
Liquid storage liquid: take 100mg Tianhusui Yugan tablets, be dissolved in 5ml dehydrated alcohol, 0.2 ��m of filter filters, 500 �� ldoff pipe packing ,-20 DEG C of storages, and 0.2 ��m of filter filters the use of dehydrated alcohol in order to control group simultaneously.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Foetal calf serum (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC03(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); 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
Lay card inverted microscope (Germany Leica model: DMIL); Visible-UV-light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); C02Incubator (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/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); Desk-top electric drying oven with forced convection (the accurate experimental installation company in Shanghai model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (above Hai'an booth scientific instrument factory model: KA-1000); 0.2 ��m of filter (MILLIPORE model: SLGP033RB); 1cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, transfer pipet, Tips are some.
2. experimental technique
1) Du-145 cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when cell grows to logarithmic phase, collecting cell, abandons nutrient solution, and PBS gently washes 3 times, add 3ml0.25% trypsinase-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml perfect medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment 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 into cell culture incubator (37 DEG C, 5%C02) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add Tianhusui Yugan tablets 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 thieving paper, and every hole adds 200 �� l dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on shaking table, crystallisate is fully dissolved. The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (not adding cell, only add nutrient solution) is set simultaneously, control wells (the medicine dissolution medium of cell, same concentrations, nutrient solution, MTT, dimethyl sulfoxide (DMSO)), often the multiple hole of group setting 6.
7) inhibiting rate of cell is represented by result with medicine: cell proliferation inhibiting rate (%)=(control wells OD value-dosing holes OD value), control wells OD value �� 100%. Experiment repeats 3 times.
3. statistical treatment
Adopting 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 control group, when dosage reaches 5mg/ml, to Du-145 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 Tianhusui Yugan tablets is to Du-145 cell inhibitory effect influence research (X �� SD)
Group Drug level (mg/ml) Inhibiting rate (%)
Control group 0 0
1 5 8.59��2.28
2 10 16.39��4.06*
3 15 22.34��6.89**
4 20 28.34��9.05**
Note: compare with control group, * P < 0.01; * P < 0.001.
5. experiment conclusion
The Tianhusui Yugan tablets of the present invention can suppress Du-145 cell proliferation, reduces the cell growing number of Du-145 cell, and this effect is dose-dependently.

Claims (2)

1. Tianhusui Yugan tablets suppresses the application in prostate cancer cell Du-145 cell proliferation in preparation, described Tianhusui Yugan tablets is made up as bulk drug of Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, it is characterized in that, the preparation method of described Tianhusui Yugan tablets comprises the steps: to get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, joins CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 4-6%, extracting pressure 15-30MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 800-1000min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
2. Tianhusui Yugan tablets according to claim 1 suppresses the application in prostate cancer cell Du-145 cell proliferation in preparation, it is characterized in that, the preparation method of described Tianhusui Yugan tablets comprises the steps: to get Lawn Pennywort Herb 699g, Root of Candolle Pimpinella 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, joins CO2In supercritical extraction device, ethanol is as entrainment agent, and it is 5%, extracting pressure 25MPa that entrainment agent accounts for the volume percent of total extraction solvent, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 900min, obtains supercritical extract, supercritical extract is added starch, 70% ethanol particle, and dry, compressing tablet, makes 500, the heavy 0.30g of every sheet.
CN201610183033.3A 2016-03-28 2016-03-28 Application of Tianhusui Yugan tablet to preparation of cell proliferation drug for restraining prostate cancer cells Du-145 Withdrawn CN105616562A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103566023A (en) * 2013-10-29 2014-02-12 山东省立医院 Preparation method and application of toothache anti-inflammation tablets

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
范文昌等，: "《广东地产清热解毒药物大全》", 31 July 2011, 中医古籍出版社 *

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