CN105726670A - Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting oral epidermoid carcinoma cell KB proliferation - Google Patents

Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting oral epidermoid carcinoma cell KB proliferation Download PDF

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Publication number
CN105726670A
CN105726670A CN201610183053.0A CN201610183053A CN105726670A CN 105726670 A CN105726670 A CN 105726670A CN 201610183053 A CN201610183053 A CN 201610183053A CN 105726670 A CN105726670 A CN 105726670A
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preparation
yugan tablets
tianhusui yugan
tablet
yugan
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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)
    • A61K36/71—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
    • 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)
    • A61K36/23—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
    • 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)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention belongs to the technical field of traditional Chinese medicines and in particular relates to an application of a lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting oral epidermoid carcinoma cell KB proliferation and a preparation method of the lawn pennywort herb Yugan tablet. The lawn pennywort herb Yugan tablet is prepared by using 699g of lawn pennywort herb, 1398g of root of candolle pimpinella, 699g of creeping woodsorrel herb and 209g of brooklet anemone root as raw medicines and is prepared by adopting supercritical extraction, so that the content of oleanolic acid is greatly increased.

Description

Tianhusui Yugan tablets suppresses the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the preparation method that a kind of Tianhusui Yugan tablets suppresses the application in oral cavity epidermoid carcinoma cell KB 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 standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle.It is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, there is heat-clearing and toxic substances removing, effect of depressed liver-energy dispersing and function of gallbladder promoting, 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 suppressed in human mouth epidermoid carcinoma cell KB cell proliferation in preparation, also extract preparation aspect there are no Tianhusui Yugan tablets and adopt the report of supercritical extraction, and the method that traditional water decoction is boiled, technique is coarse, backward, and impurity is many, causes that patient's consumption is 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 a kind of Tianhusui Yugan tablets and suppress the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation.
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 suppresses the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation, 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%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, 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, dry, and tabletting makes 500, every tablet weight 0.30g.
Preferably, above-mentioned Tianhusui Yugan tablets suppresses the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation, 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, dry, and 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.The every tablet weight 0.30g of Tianhusui Yugan tablets adopting the inventive method to prepare, only needs 3 every time, within 1st, takes 3 times.Dose is greatly reduced when having more active component.This conclusion can pass through following it have been experienced that.
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 mobile phase；Detection wavelength is 210nm.Number of theoretical plate calculates 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, weighed weight again, 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, water-bath is concentrated into without alcohol taste, let cool, 3 (40ml are extracted with dichloromethane jolting, 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, weighed weight again, 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, water-bath is concentrated into without alcohol taste, let cool, 3 (40ml are extracted with dichloromethane jolting, 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
It is shown that the content of oleanolic acid is 3.22-6.45mg/ sheet in Tianhusui Yugan tablets of the present invention；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, when dose reduces, content of oleanolic acid improves a lot.
The studies above shows, adopts 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, dry, and 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, dry, and 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, dry, and tabletting makes 500, every tablet weight 0.30g.
After testing, in finished product, the content of oleanolic acid is 3.22mg/ sheet.
Embodiment 4: Tianhusui Yugan tablets suppresses the experimentation data of human mouth epidermoid carcinoma cell KB cell proliferation
1. experiment material
1.1 experiment cell strains
Human mouth epidermoid carcinoma cell KB 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, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagents
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137)；Hyclone (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 equipments
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 of 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) KB cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when Growth of Cells to logarithmic (log) phase, collect cell, discard culture fluid, PBS fine laundering 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 reaction, is 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 place.
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 repeats 3 times.
3. statistical disposition
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 matched group, when dosage reaches 5mg/ml, to KB 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 KB cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 9.68±3.26
2 10 18.31±5.12*
3 15 25.96±8.03**
4 20 32..52±10.18**
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
The Tianhusui Yugan tablets of the present invention can suppress KB cell proliferation, reduces the Growth of Cells number of KB cell, and this effect is dose dependent.

Claims (2)

1. Tianhusui Yugan tablets suppresses the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation, 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%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, 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, dry, and tabletting makes 500, every tablet weight 0.30g.
2. Tianhusui Yugan tablets according to claim 1 suppresses the application in oral cavity epidermoid carcinoma cell KB cell proliferation in preparation, 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, dry, and tabletting makes 500, every tablet weight 0.30g.
CN201610183053.0A 2016-03-28 2016-03-28 Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting oral epidermoid carcinoma cell KB proliferation Withdrawn CN105726670A (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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Application publication date: 20160706