CN105816536A - Application of lawn pennywort herb liver-healing tablets in preparation of drug for inhibiting cell proliferation of cerebral tumor cells SF767 - Google Patents
Application of lawn pennywort herb liver-healing tablets in preparation of drug for inhibiting cell proliferation of cerebral tumor cells SF767 Download PDFInfo
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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
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- 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)
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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)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
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- 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
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- 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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- 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 medicine, and particularly relates to application of lawn pennywort herb liver-healing tablets in preparation of a drug for inhibiting cell proliferation of cerebral tumor cells SF767 and a preparation method of the lawn pennywort herb liver-healing tablets.The lawn pennywort herb liver-healing tablets are prepared from 699 g of lawn pennywort herb, 1,398 g of herba pimpinellae, 699 g of creeping woodsorrel herb and 209 g of brooklet anemone roots.The lawn pennywort herb liver-healing tablets are prepared by means of supercritical extraction, and the content of oleanolic acid is greatly increased.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the application in preparation suppression Tumor cells SF767 cell proliferation of a kind of Tianhusui Yugan tablets and the preparation method of Tianhusui Yugan tablets.
Background technology
Tianhusui Yugan tablets standard No. WS-10213(ZD-0213)-2002, it is recorded in country's standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle.Being made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, have heat-clearing and toxic substances removing, effect of depressed liver-energy dispersing and function of gallbladder promoting, for the acute hepatitis, chronic hepatitis caused by dampness-heat in the liver and gallbladder.
In prior art, not yet there is the Tianhusui Yugan tablets report in the application in preparation suppression SHG44 cells cell SF767 cell proliferation, also there are no Tianhusui Yugan tablets and extract the report of preparation aspect employing 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, 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 Tumor cells SF767 cell proliferation of a kind of Tianhusui Yugan tablets.
Further object is that the preparation method that a kind of Tianhusui Yugan tablets is provided.
It is an object of the invention to by following scheme realization:
Tianhusui Yugan tablets application in preparation suppression Tumor cells SF767 cell proliferation, described Tianhusui Yugan tablets is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, the preparation method of described Tianhusui Yugan tablets comprises the steps: to take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 800-1000min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
Preferably, the application in preparation suppression Tumor cells SF767 cell proliferation of the above-mentioned Tianhusui Yugan tablets, the preparation method of described Tianhusui Yugan tablets comprises the steps: to take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 900min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
In prior art, Tianhusui Yugan tablets be administered orally, one time 6,3 times on the one.Tianhusui Yugan tablets dosage is big.Every tablet weight 0.30g of Tianhusui Yugan tablets using the inventive method to be prepared as, the most only needs 3, 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 oleanolic acid in Tianhusui Yugan tablets prepared by test one, distinct methods
L, instrument and reagent Tianhusui Yugan tablets of the present invention: prepare by embodiment 1 method, uses 3005g crude drug, extracted makes 500, every tablet weight 0.30g.Former Tianhusui Yugan tablets, according to WS-10213(ZD-0213) prepared by-2002 standard methods.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 the 4th note on the use 15 of version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Acetonitrile-0.1% phosphoric acid (80:20) is flowing phase；Detection wavelength is 210nm.Number of theoretical plate is calculated by oleanolic acid peak should be not less than 3000.
It is appropriate that the preparation precision of reference substance solution weighs oleanolic acid reference substance, adds methanol and makes every 1ml solution containing 0.2mg, to obtain final product.
The preparation of the Tianhusui Yugan tablets need testing solution of the present invention takes the Tianhusui Yugan tablets of the present invention, finely ground, take the amount of 0.75g(2.5 sheet), accurately weighed, put in tool plug conical flask, accurate addition ethanol 25ml, close plug, weighed weight, it is heated to reflux 1 hour, let cool, the most weighed weight, the weight of less loss is supplied with ethanol, shake up, filter, precision measures subsequent filtrate 10ml, add 20% hydrochloric acid solution 5ml, it is heated to reflux 2.5 hours, add water 30ml, it is concentrated into without alcohol taste in water-bath, let cool, 3 (40ml are extracted with dichloromethane shaking, 30ml, 30ml), combined dichloromethane extracting solution, it is evaporated, residue adds methanol makes dissolving in right amount, it is transferred in 10ml measuring bottle, add methanol dilution to scale, shake up, obtain.
The preparation of reference product need testing solution takes the Tianhusui Yugan tablets 20g of comparison, finely ground, take the amount of 1.5g(5 sheet), accurately weighed, put in tool plug conical flask, accurate addition ethanol 25ml, close plug, weighed weight, it is heated to reflux 1 hour, let cool, the most weighed weight, the weight of less loss is supplied with ethanol, shake up, filter, precision measures subsequent filtrate 10ml, add 20% hydrochloric acid solution 5ml, it is heated to reflux 2.5 hours, add water 30ml, it is concentrated into without alcohol taste in water-bath, let cool, 3 (40ml are extracted with dichloromethane shaking, 30ml, 30ml), combined dichloromethane extracting solution, it is evaporated, residue adds methanol makes dissolving in right amount, it is transferred in 10ml measuring bottle, add methanol dilution to scale, shake up, obtain.
Algoscopy precision respectively draws reference substance solution and the Tianhusui Yugan tablets need testing solution of the present invention, each 10 μ l of reference product need testing solution, injects chromatograph of liquid, 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 2-4 times that content of oleanolic acid is former 6 content of tablet of consumption 3, in the case of dose reduces, content of oleanolic acid improves a lot.
The studies above shows, uses Tianhusui Yugan tablets prepared by preparation method of the present invention, and active constituent content is significantly larger than WS-10213(ZD-0213) Tianhusui Yugan tablets 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 Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 900min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of oleanolic acid is 6.45mg/ sheet.
Embodiment 2
Take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 1000min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of oleanolic acid is 4.98mg/ sheet.
Embodiment 3
Take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of oleanolic acid is 3.22mg/ sheet.
Embodiment 4: the experimentation data of Tianhusui Yugan tablets suppression SHG44 cells cell SF767 cell proliferation
1. experiment material
1.1 experiment cell strains
SHG44 cells cell SF767 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Tianhusui Yugan tablets of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh 100mg Tianhusui Yugan tablets, is dissolved in 5ml dehydrated alcohol, and 0.2 μm filter filters, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and 0.2 μm 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 μm 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) SF767 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 μ 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 Tianhusui Yugan tablets solution, continue to cultivate 24h.
4) add 20 μ 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 μ 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 SF767 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 Tianhusui Yugan tablets is to SF767 cell inhibitory effect influence research (X ± SD)
| Group | Drug level (mg/ml) | Suppression ratio (%) |
| Matched group | 0 | 0 |
| 1 | 5 | 12.24±2.36 |
| 2 | 10 | 23.05±4.59* |
| 3 | 15 | 34.68±7.21** |
| 4 | 20 | 40.69±9.89** |
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
The Tianhusui Yugan tablets of the present invention can suppress SF767 cell proliferation, reduces the cell growing number of SF767 cell, and this effect is dose dependent.
Claims (2)
1. Tianhusui Yugan tablets application in preparation suppression Tumor cells SF767 cell proliferation, described Tianhusui Yugan tablets is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 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 take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 800-1000min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
The Tianhusui Yugan tablets the most according to claim 1 application in preparation suppression Tumor cells SF767 cell proliferation, it is characterized in that, the preparation method of described Tianhusui Yugan tablets comprises the steps: to take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, 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 900min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, is dried, tabletting, makes 500, every tablet weight 0.30g.
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| CN201610183050.7A CN105816536A (en) | 2016-03-28 | 2016-03-28 | Application of lawn pennywort herb liver-healing tablets in preparation of drug for inhibiting cell proliferation of cerebral tumor cells SF767 |
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| CN201610183050.7A CN105816536A (en) | 2016-03-28 | 2016-03-28 | Application of lawn pennywort herb liver-healing tablets in preparation of drug for inhibiting cell proliferation of cerebral tumor cells SF767 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200133541A (en) * | 2019-05-20 | 2020-11-30 | 고려대학교 세종산학협력단 | Composition for preventing or treating cancer comprising extracts of Hydrocotyle Umbellata |
Citations (1)
| 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 |
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| 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)
| 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 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200133541A (en) * | 2019-05-20 | 2020-11-30 | 고려대학교 세종산학협력단 | Composition for preventing or treating cancer comprising extracts of Hydrocotyle Umbellata |
| KR102223039B1 (en) * | 2019-05-20 | 2021-03-03 | 고려대학교 세종산학협력단 | Composition for preventing or treating cancer comprising extracts of Hydrocotyle Umbellata |
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Application publication date: 20160803 |