CN105726668A - Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting small cell lung cancer cell LTEP-P proliferation - Google Patents

Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting small cell lung cancer cell LTEP-P proliferation Download PDF

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Publication number
CN105726668A
CN105726668A CN201610183025.9A CN201610183025A CN105726668A CN 105726668 A CN105726668 A CN 105726668A CN 201610183025 A CN201610183025 A CN 201610183025A CN 105726668 A CN105726668 A CN 105726668A
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preparation
yugan
ltep
tablet
radix
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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)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (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 small cell lung cancer cell LTEP-P 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 is at preparation suppression small cell lung cancer cell LTEP-P cell proliferation medicine Application in thing
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Tianhusui Yugan tablets thin at preparation suppression small cell lung cancer Application in born of the same parents' LTEP-P cell proliferation 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.It is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, has 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, Tianhusui Yugan tablets is not yet had to increase at preparation suppression human small cell lung carcinoma cell LTEP-P cell Grow the report of application in medicine, also there are no Tianhusui Yugan tablets and extract preparation aspect and use the report of supercritical extraction, and The method that traditional water decoction is boiled, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, it has not been convenient to take, have a strong impact on This product is 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 at preparation suppression small cell lung cancer cell Application in LTEP-P cell proliferation.
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 small cell lung cancer cell LTEP-P cell proliferation, described Herba Hydrocotyles YUGAN sheet is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, and described sky is recklessly The preparation method of Sui YUGAN sheet 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 entrainer accounts for the percent by volume of total extractant and is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 800- 1000min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, makes 500 Sheet, every tablet weight 0.30g.
Preferably, above-mentioned Tianhusui Yugan tablets is in preparation suppression small cell lung cancer cell LTEP-P cell proliferation Application, the preparation method of described Tianhusui Yugan tablets comprises the steps: to take Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, jealous woman slurry Grass 699g, Radix Anemones Rivularis 209g, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for total extractant Percent by volume is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 900min, Supercritical extract, adds starch by supercritical extract, 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.Use Every tablet weight 0.30g of Tianhusui Yugan tablets that the inventive method is prepared as, the most only needs 3, within 1st, takes 3 times.More having Dose is greatly reduced under conditions of 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 system Become 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；METTLER AE240 electronic analytical balance；(China's medicine is raw for oleanolic acid reference substance Tetramune examines and determine institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia the 4th note on the use 15 of version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Acetonitrile-0.1% phosphorus 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 containing 0.2mg's Solution, 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, takes The amount of 0.75g(2.5 sheet), accurately weighed, put in tool plug conical flask, the accurate ethanol 25ml that adds, close plug, weighed weight, heating Reflux 1 hour, let cool, more weighed weight, to supply the weight of less loss with ethanol, shake up, filter, precision measures subsequent filtrate 10ml, Adding 20% hydrochloric acid solution 5ml, be heated to reflux 2.5 hours, add water 30ml, water-bath is concentrated into without alcohol taste, lets cool, uses dichloromethane 3 times (40ml, 30ml, 30ml), combined dichloromethane extracting solution are extracted in shaking, are evaporated, and residue adds methanol makes dissolving in right amount, transfer To 10ml measuring bottle, add methanol dilution to scale, shake up, to obtain final product.
The preparation of reference product need testing solution takes the Tianhusui Yugan tablets 20g of comparison, finely ground, takes 1.5g(5 sheet Amount), accurately weighed, put in tool plug conical flask, accurate addition ethanol 25ml, close plug, weighed weight, it is heated to reflux 1 hour, puts Cold, more weighed weight, to supply the weight of less loss with ethanol, shake up, filter, precision measures subsequent filtrate 10ml, adds 20% hydrochloric acid molten Liquid 5ml, is heated to reflux 2.5 hours, and add water 30ml, water-bath is concentrated into without alcohol taste, lets cool, and extracts 3 times with dichloromethane shaking (40ml, 30ml, 30ml), combined dichloromethane extracting solution, it is evaporated, residue adds methanol makes dissolving in right amount, is transferred to 10ml measuring bottle In, add methanol dilution to scale, shake up, to obtain final product.
Algoscopy respectively precision is drawn the Tianhusui Yugan tablets need testing solution of reference substance solution and the present invention, is compareed product The each 10 μ l of product need testing solution, inject chromatograph of liquid, measure, 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 former Herba Hydrocotyles is more In liver slice, the content of oleanolic acid is 0.81mg/ sheet, and the content of oleanolic acid each serving consumption 3 is former 6 content of tablet 2-4 times, 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 the highest In 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, in order to those skilled in the art knows more about The present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, all above-mentioned based on the present invention The technology that content is realized belongs to the scope of the present invention.
Embodiment 1
Take 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, CO2 Flow 2ml/g crude drug min, extraction time 900min, obtain supercritical extract, supercritical extract added starch, 70% second Alcohol 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, CO2 Flow 1ml/g crude drug min, extraction time 1000min, obtain supercritical extract, supercritical extract added starch, and 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, CO2 Flow 3ml/g crude drug min, extraction time 800min, obtain supercritical extract, supercritical extract added starch, 70% second Alcohol 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 human small cell lung carcinoma cell LTEP-P cell proliferation
1. experiment material
1.1 experiment cell strains
Human small cell lung carcinoma cell LTEP-P 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 m filter filters, and 500 Ldoff pipe subpackage ,-20 DEG C of storages, 0.2 m filter filters dehydrated alcohol in case matched group is used simultaneously.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061 Lot.No.758137)；Hyclone (Hangzhoupro, sky, Zhejiang biotechnology Company limited Lot.No.100419)；NaHC03(the long hundred million chemical reagent company limited Cat.No.11810-033 in Shanghai Lot.No. 1088387)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)；Penicillin G Sodium Salt(AMRESCO company 1)；Streptomycin Sulfate(AMRESCO)；(Zibo Ya Dulan economy and trade is limited for dehydrated alcohol Company)；MTT (Biosharp lot number: 0793): PBS(laboratory autogamy)；
1.4 experiment equipment
Lycra inverted microscope (Germany Leica model: DMIL)；Visible-ultraviolet light microwell plate detector (MD company type of the U.S. Number: SPECTRAMAX 190)；C02Incubator (FORMA model: 3111)；Super-clean bench (safe and sound company of Su Jing group manufacture model: SW-CJ-ZFD)；Pure water instrument (Sprlng company of U.S. model: S/N 020579)；Accurate pipettor (Gilson Inc of France type Number: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；(Japan SANYO is public for full-automatic high-pressure autoclave Department model: MLS-3020)；Table electrothermal air dry oven (Shanghai precision experimental facilities company model: DHG9123A)；Refrigerator (Siemens Company's model: KG18V21TI)；Liquid nitrogen container (CBS model: 2001)；Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai Model: KA-1000)；0.2 m filter (MILLIPORE model: SLGP033RB)；1cm culture dish (NEST company), 96 holes are cultivated Plate (NEST company)；Cell counting count board；Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) LTEP-P cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when cell grows During to logarithmic (log) phase, collecting cell, discard culture fluid, PBS rinses 3 times, addition 3ml 0.25% trypsin-0.04%EDTA, and 37 After DEG C digestion 2min, it is added thereto in 5ml complete medium and react, blowing and beating after cell and proceeded in centrifuge tube, 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, and culture plate is put in cell culture incubator (37 DEG C, 5%C02) cellar culture.
3) according to cell growth status, general long to 50%-70%, add Tianhusui Yugan tablets solution, continue to cultivate 24h.
4) add 20 l MTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 l dimethyl sulfoxide, puts shaking table Upper low-speed oscillation 10min, makes crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm.
6) arranging background (being not added with cell, only add culture fluid), (cell, the medicine dissolution of same concentrations are situated between control wells simultaneously Matter, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes.
7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD Value), control wells OD value × 100%.Experiment is repeated 3 times.
3. statistical disposition
Using the correlation analysis in Microsoft Excel 2007 software and Student t inspection, data are with mean ± S.D. Represent.
4. experimental result
Statistical result showed after mtt assay experiment, compares with matched group, when dosage reaches 5mg/ml, to LTEP-P cell proliferation Suppressing variant (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), when dosage reaches 15-20mg/ Pole significant difference (P < 0.001) is had during ml.
Table 1 Tianhusui Yugan tablets is to LTEP-P 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 LTEP-P cell proliferation, reduces the cell growing number of LTEP-P cell, This effect is dose dependent.

Claims (2)

1. Tianhusui Yugan tablets application in preparation suppression small cell lung cancer cell LTEP-P cell proliferation, described sky is recklessly Sui YUGAN sheet is made up as crude drug of Herba Hydrocotyles 699g, Radix Pimpinellae Candolleanae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, its feature Being, 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, join CO2In supercritical extraction device, ethanol is as entrainer, and entrainer accounts for the body of total extractant Long-pending percentage ratio is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 800-1000min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, system Become 500, every tablet weight 0.30g.
2. according to the Tianhusui Yugan tablets described in claim l at preparation suppression small cell lung cancer cell LTEP-P cell proliferation medicine Application in thing, it is characterised in that the preparation method of described Tianhusui Yugan tablets comprises the steps: to take Herba Hydrocotyles 699g, Fructus Pruni Leaf Radix Saposhnikoviae 1398g, Herba Oxalidis Corniculatae 699g, Radix Anemones Rivularis 209g, join CO2In supercritical extraction device, ethanol, as entrainer, is carried secretly It is 5% that agent accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, Extraction time 900min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, is dried, tabletting, Make 500, every tablet weight 0.30g.
CN201610183025.9A 2016-03-28 2016-03-28 Application of lawn pennywort herb Yugan tablet to preparation of drugs for inhibiting small cell lung cancer cell LTEP-P proliferation Withdrawn CN105726668A (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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